相关申请的交叉引用Cross References to Related Applications
本申请要求2015年2月8日提交的美国临时专利申请第62/113,480号的权益和优先权以及2015年4月7日提交的中国专利申请第201510160468.1号的权益和优先权，上述美国临时专利申请和中国专利申请的全部内容通过引用并入本文。This application claims the benefit and priority of U.S. Provisional Patent Application No. 62/113,480, filed February 8, 2015, and Chinese Patent Application No. 201510160468.1, filed April 7, 2015, the aforementioned U.S. Provisional Patent The entire contents of the application and the Chinese patent application are hereby incorporated by reference.
技术领域technical field
本发明涉及包含诸如吲哚菁绿(ICG)之类的染料的脂质纳米颗粒的制备和使用，用于增强吲哚菁绿近红外荧光成像的强度和分辨率，并用于体内正常或异常组织和细胞的显影及诊断。The present invention relates to the preparation and use of lipid nanoparticles comprising dyes such as indocyanine green (ICG) for enhancing the intensity and resolution of near-infrared fluorescence imaging of indocyanine green and for normal or abnormal tissues in vivo And cell development and diagnosis.
背景技术Background technique
为了对疾病进行有效治疗和诊断，能够对受影响的器官进行成像是比较有利的。目前在本领域中已有多种被广泛使用的成像技术；然而，这些技术往往不具有通用性或者缺乏能够识别脉管系统或淋巴系统的对比度。一种研究这些系统的新兴技术是光成像技术，其被称为近红外荧光(NIRF)成像(～700-900nm)，该技术因不涉及电离辐射而具有非常高的安全性，具有最小的来自血液和组织自体荧光(～500-600nm)(即，背景信号/荧光)的干扰，并且是无创的。荧光成像方法涉及使用光线激发荧光染料并且检测从染料中发射出的荧光，该荧光成像方法被广泛用于各种类型的生物成像。该方法用于血管造影术，并且可用于手术过程中对血管功能和/或肿瘤转移的评估。不像其他技术那样，NIRF成像还具有使淋巴系统成像的能力，这为临床医师提供了关于患者体内淋巴结构和功能的重要信息。尽管如此，NIRF成像由于无法使深层组织成像(因光散射问题仅能使～1cm深度的组织成像)而面临很多问题。For effective treatment and diagnosis of disease, it is advantageous to be able to image the affected organ. There are several imaging techniques that are widely used in the art; however, these techniques are often not versatile or lack the contrast to identify the vasculature or lymphatic system. An emerging technique for studying these systems is optical imaging, known as near-infrared fluorescence (NIRF) imaging (~700-900nm), which is very safe since it does not involve ionizing radiation, with minimal radiation from Interferes with blood and tissue autofluorescence (~500-600nm) (ie, background signal/fluorescence) and is noninvasive. Fluorescence imaging methods, which involve exciting fluorescent dyes with light and detecting fluorescent light emitted from the dyes, are widely used in various types of biological imaging. This method is used in angiography and can be used in the assessment of vascular function and/or tumor metastasis during surgery. Unlike other techniques, NIRF imaging also has the ability to image the lymphatic system, which provides clinicians with important information about lymphatic structure and function in patients. Nevertheless, NIRF imaging faces many problems due to its inability to image deep tissues (only ~1 cm deep tissue can be imaged due to light scattering problems).
ICG是临床上NIRF成像中所使用的染料(具有820nm发射波长)并且是美国食品与药品管理局(FDA)和欧洲药物管理局(EMA)唯一批准的用于人体的NIRF分子。ICG被批准用于心输出量、肝脏功能和肝脏血流检测，以及眼部血管造影术。ICG存在着大量的标签外和以研究为目的的使用情况，包括液体填充的解剖学结构(例如，血液、脑脊液、淋巴或尿液)的可视化，或作为对比剂用于血管、肾脏或其它排泄途径的显影(1)。在水性环境中，ICG分子聚集并且ICG荧光强度容易衰减(降低整体荧光强度)(2-5)。在血液中，ICG与血浆蛋白结合，部分且暂时地改善了其荧光强度，但是最终仍然会与血浆蛋白解离进入水相环境中被降解。在体内，ICG荧光强度和持续时间可随血浆蛋白和脂蛋白浓度的波动以及个体之间的差异而发生变化。ICG is the dye used clinically in NIRF imaging (with an emission wavelength of 820 nm) and is the only NIRF molecule approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for use in humans. ICG is approved for cardiac output, liver function, and liver blood flow testing, as well as ocular angiography. Numerous off-label and research-oriented uses of ICG exist, including visualization of fluid-filled anatomical structures (eg, blood, cerebrospinal fluid, lymph, or urine), or as a contrast agent for vascular, renal, or other excretory Visualization of pathways (1). In an aqueous environment, ICG molecules aggregate and ICG fluorescence intensity tends to decay (reduce overall fluorescence intensity) (2-5). In blood, ICG binds to plasma proteins, partially and temporarily improving its fluorescence intensity, but eventually dissociates from plasma proteins and enters the aqueous environment for degradation. In vivo, ICG fluorescence intensity and duration can vary with fluctuations in plasma protein and lipoprotein concentrations and with interindividual variability.
最近关于具有多种不同的生理化学性质的ICG和脂质体的报道描述了将ICG包裹在脂质体内的资源密集型制备步骤(≥4个步骤)(8-12)。然而，这些具有不同组成的脂质体是在没有完全理解ICG和脂质之间的相互作用的条件下制备的。在一些条件下，当ICG包裹在脂质体的水性腔室内时，ICG可能部分或通过物理键的形式与脂类分子膜发生相互作用。根据这些方法结合的脂质-ICG以及脂质-ICG组合物是不完全且不稳定的。这些制剂不以使ICG插入或嵌入脂质中为目的，从而导致ICG脂质体组合物无论在产品稳定性还是在真正有效被稳定组装的ICG分子数目上均存在着很大的变异度，并且在其用作成像产品方面的结果也各不相同。而且，哪怕仅一部分ICG可能被包裹进入脂质体的水性腔室，也需要在制备过程中除去游离的ICG，该步骤会增加潜在的污染，并且由于损耗和分离过程而增加成本。Recent reports on ICG and liposomes with diverse physiochemical properties describe resource-intensive manufacturing steps (≥4 steps) for encapsulation of ICG within liposomes (8-12). However, these liposomes with different compositions were prepared without a complete understanding of the interaction between ICG and lipids. Under some conditions, when ICG is encapsulated in the aqueous chamber of liposomes, ICG may interact with the lipid molecular membrane partially or through physical bonds. Lipid-ICG and lipid-ICG compositions conjugated according to these methods are incomplete and unstable. These formulations do not have the purpose of intercalating or intercalating ICG into lipids, resulting in large variability in ICG liposome compositions both in product stability and in the number of ICG molecules that are actually effectively and stably assembled, and Results have also been mixed regarding its use as an imaging product. Moreover, even though only a portion of ICG may be encapsulated into the aqueous compartment of liposomes, free ICG needs to be removed during preparation, a step that increases potential contamination and costs due to loss and isolation procedures.
本领域亟需对ICG递送系统进行改善。There is a great need in the art for improvements in ICG delivery systems.
发明内容Contents of the invention
一方面，本发明提供一种组合物，其包含(i)含有脂质膜和水性核的脂质纳米颗粒，以及(ii)多个吲哚菁绿(ICG)，其中，ICG中的一个或多个嵌入所述脂质膜中。In one aspect, the present invention provides a composition comprising (i) a lipid nanoparticle comprising a lipid membrane and an aqueous core, and (ii) a plurality of indocyanine green (ICG), wherein one or Multiples are embedded in the lipid membrane.
在一些实施方式中，脂质分子包含1,2-二硬脂酰基-sn-丙三基-3-磷酸胆碱(1,2-distearoyl-sn-glycero-3-phosphocholine，DSPC)和1,2-二硬脂酰基-sn-丙三基-3-磷酸乙醇胺-N-甲氧基-聚乙二醇-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-polyethyleneglycol-2000，DSPEmPEG2000)。In some embodiments, the lipid molecule comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC) and 1, 2-Distearoyl-sn-propanetriyl-3-phosphoethanolamine-N-methoxy-polyethylene glycol-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-polyethyleneglycol -2000, DSPEmPEG2000).
在一些实施方式中，至少约95％的ICG嵌入脂质膜中。在一些实施方式中，至少约99％的ICG嵌入脂质膜中。在一些实施方式中，基本约100％的ICG嵌入脂质膜中。In some embodiments, at least about 95% of the ICG is embedded in the lipid membrane. In some embodiments, at least about 99% of the ICG is embedded in the lipid membrane. In some embodiments, substantially about 100% of the ICG is embedded in the lipid membrane.
在一些实施方式中，低于5％的ICG包裹在水性核中。在一些实施方式中，低于1％的ICG包裹在水性核中。在一些实施方式中，没有可检测量的ICG包裹在水性核中。In some embodiments, less than 5% of the ICG is encapsulated in the aqueous core. In some embodiments, less than 1% of the ICG is encapsulated in the aqueous core. In some embodiments, no detectable amount of ICG is encapsulated in the aqueous core.
在一些实施方式中，脂质纳米颗粒悬浮于盐组合物中，所述盐组合物包含生物相容性缓冲液，例如，pH为5-8且渗透压为约303mOSM的生物相容性缓冲液。这些缓冲液的实例包括生理盐水，林格氏溶液，5％葡萄糖水溶液或0.9％NaCl和约20mM NaHCO3的缓冲液。In some embodiments, the lipid nanoparticles are suspended in a saline composition comprising a biocompatible buffer, e.g., a biocompatible buffer with a pH of 5-8 and an osmolarity of about 303 mOSM . Examples of these buffers include physiological saline, Ringer's solution, 5% dextrose in water, or a buffer of 0.9% NaCl and about 20 mMNaHCO3 .
在一些实施方式中，纳米颗粒的平均尺寸是50nm至100nm。In some embodiments, the nanoparticles have an average size of 50 nm to 100 nm.
在一些实施方式中，纳米颗粒表面带有负电荷。In some embodiments, the surface of the nanoparticles is negatively charged.
在一些实施方式中，纳米颗粒在血清中稳定，并且，在25℃下，在热灭活的大鼠血清中六小时后保留所述纳米颗粒的初始荧光的约90％至约100％。In some embodiments, the nanoparticles are stable in serum and retain about 90% to about 100% of the initial fluorescence of the nanoparticles after six hours in heat-inactivated rat serum at 25°C.
在一些实施方式中，所述纳米颗粒的荧光强度是水性溶液中游离ICG的荧光强度的4倍至5倍。In some embodiments, the fluorescence intensity of the nanoparticles is 4 to 5 times that of free ICG in aqueous solution.
在一些实施方式中，在4℃下储存0至约300天之后，所述纳米颗粒的荧光强度是水性溶液中游离ICG的荧光强度的4倍至100,000倍。In some embodiments, the nanoparticle has a fluorescence intensity that is 4 to 100,000 times that of free ICG in aqueous solution after storage at 4°C for 0 to about 300 days.
在一些实施方式中，所述组合物在低于约0.5mg/kg体重的ICG剂量条件下有效。在一些实施方式中，所述组合物在约0.01mg/kg体重的ICG剂量条件下有效。In some embodiments, the composition is effective at an ICG dosage of less than about 0.5 mg/kg body weight. In some embodiments, the composition is effective at an ICG dosage of about 0.01 mg/kg body weight.
另一方面，本发明提供一种试剂盒，所述试剂盒包含本文提供的组合物/LNP以及任选地包含如何使用所述试剂盒的说明书。In another aspect, the invention provides a kit comprising a composition/LNP provided herein and optionally instructions how to use the kit.
又一方面，本发明提供一种使受治者体内的组织或器官成像的方法，所述方法包括：将适量的本文提供的组合物给药于需要进行成像的受治者，获取所述受治者的组织或器官的图像。In yet another aspect, the present invention provides a method for imaging tissues or organs in a subject, the method comprising: administering an appropriate amount of the composition provided herein to a subject in need of imaging, and obtaining the image of the subject's tissue or organ.
在一些实施方式中，所述组合物的给药途径选自全身给药和局部给药，其中，所述全身给药包括血管内注射，所述局部给药包括皮下注射、皮内注射、粘膜下注射、浆膜下注射、肿瘤内注射、肌肉内注射、口服、经鼻给药和肿瘤内注射。在本发明优选的实施方式中，在通过血管内注射的方式给药的情况下，所述组合物的量等于约0.01mg/kg体重ICG剂量至约0.5mg/kg体重ICG剂量。在局部给药本发明的组合物的情况下，所述组合物的单点注射量等于约0.1ng至约0.1mg ICG剂量。In some embodiments, the route of administration of the composition is selected from systemic administration and local administration, wherein the systemic administration includes intravascular injection, and the local administration includes subcutaneous injection, intradermal injection, mucosal injection, etc. Sub-injection, subserosal injection, intratumoral injection, intramuscular injection, oral, nasal administration and intratumoral injection. In a preferred embodiment of the present invention, in the case of administration by intravascular injection, the amount of said composition is equal to about 0.01 mg/kg body weight ICG dose to about 0.5 mg/kg body weight ICG dose. In the case of topical administration of a composition of the invention, a single point injection of the composition is equivalent to a dose of about 0.1 ng to about 0.1 mg ICG.
在一些实施方式中，所述组织或器官选自：淋巴管、二级淋巴组织、血管、诸如动脉粥样硬化之类的内皮病变，癌症/肿瘤、诸如在炎症位点的三级淋巴组织、肝脏、胆管、胆囊和肠道。In some embodiments, the tissue or organ is selected from the group consisting of lymphatic vessels, secondary lymphoid tissue, blood vessels, endothelial lesions such as atherosclerosis, cancer/tumor, tertiary lymphoid tissue such as at sites of inflammation, Liver, bile ducts, gallbladder and intestines.
再一方面，本发明提供一种制备纳米颗粒的方法，所述方法包括：(a)在有机溶剂中混合吲哚菁绿(ICG)和脂质分子；(b)蒸发所述有机溶剂以形成包含所述脂质分子和ICG的薄膜，以及(c)使用缓冲盐水水合所述薄膜并减小颗粒尺寸。In yet another aspect, the present invention provides a method of preparing nanoparticles, the method comprising: (a) mixing indocyanine green (ICG) and lipid molecules in an organic solvent; (b) evaporating the organic solvent to form a film comprising the lipid molecules and ICG, and (c) using buffered saline to hydrate the film and reduce particle size.
在一些实施方式中，所述脂质分子包括1,2-二硬脂酰基-sn-丙三基-3-磷酸胆碱(DSPC)和1,2-二硬脂酰基-sn-丙三基-3-磷酸乙醇胺-N-甲氧基-聚乙二醇-2000(DSPEmPEG2000)。In some embodiments, the lipid molecule comprises 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glyceryl - 3-Phosphoethanolamine-N-methoxy-polyethylene glycol-2000 (DSPEmPEG2000).
在一些实施方式中，所述有机溶剂包括乙醇、甲醇、氯仿、乙酸乙酯或DMSO。In some embodiments, the organic solvent includes ethanol, methanol, chloroform, ethyl acetate, or DMSO.
在一些实施方式中，所述缓冲盐水包含约0.9％NaCl和约20mM NaHCO3。In some embodiments, the buffered saline comprises about 0.9% NaCl and about 20 mM NaHCO3 .
在一些实施方式中，所述方法不包括过滤步骤，纯化步骤或分离步骤或者它们的组合。In some embodiments, the method does not include a filtration step, a purification step, or an isolation step, or a combination thereof.
再一方面，本发明提供一种含吲哚菁绿的颗粒，所述颗粒包含吲哚菁绿以及含有脂质膜和水性核的颗粒，其中，所述吲哚菁绿嵌入所述脂质膜中。In yet another aspect, the present invention provides an indocyanine green-containing particle comprising indocyanine green and a particle comprising a lipid membrane and an aqueous core, wherein the indocyanine green is embedded in the lipid membrane middle.
在一些实施方式中，基本上100％的吲哚菁绿嵌入所述脂质膜中。在一些实施方式中，在所述水性核中没有可检测量的吲哚菁绿。In some embodiments, substantially 100% of the indocyanine green is embedded in the lipid membrane. In some embodiments, there is no detectable amount of indocyanine green in the aqueous core.
另一方面，本发明提供一种用于光成像的对比剂，所述对比剂包含本文提供的含吲哚菁绿的颗粒和用于分散所述含吲哚菁绿的颗粒的分散介质。In another aspect, the present invention provides a contrast agent for photoimaging, the contrast agent comprising the indocyanine green-containing particles provided herein and a dispersion medium for dispersing the indocyanine green-containing particles.
附图说明Description of drawings
结合下文列出的示例性实施方式的具体描述可更好地理解本发明的特点和优势，示例性的实施方式基于本发明的基本构思，其中涉及的附图说明如下：The characteristics and advantages of the present invention can be better understood in conjunction with the specific description of the exemplary embodiments listed below. The exemplary embodiments are based on the basic concept of the present invention, wherein the accompanying drawings are described as follows:
图1显示了三步制备过程的一种实例的示意图。Figure 1 shows a schematic diagram of one example of the three-step preparation process.
图2显示了通过肌肉组织检测的ICG-LNP的荧光强度与游离ICG的荧光强度比较。(A)填充了30μM游离ICG(顶部)和ICG-LNP(底部)的毛细管的NIR荧光图像强度。(B)图A中填充了不同ICG制剂的两个相同的毛细管的白光图像。(C)重叠于填充有游离ICG(顶部)或ICG-LNP(底部)且放置于鸡胸组织方块下0.5cm、1.0cm和1.5cm(分别从左至右)的毛细管的可见光图片上的NIR荧光图像。(D)图C中的鸡胸组织方块的可见光图像。(E)重叠于放置在鸡胸组织方块下0.5cm、1.0cm和1.5cm的ICG-LNP毛细管的可见光背景上的NIR荧光图像。(F)组织方块厚度的侧视图。Figure 2 shows the fluorescence intensity of ICG-LNP detected through muscle tissue compared to that of free ICG. (A) NIR fluorescence image intensity of capillaries filled with 30 μM free ICG (top) and ICG-LNP (bottom). (B) White light images of two identical capillaries in panel A filled with different ICG formulations. (C) NIR fluorescence superimposed on visible light images of capillaries filled with free ICG (top) or ICG-LNP (bottom) and placed 0.5 cm, 1.0 cm, and 1.5 cm below chicken breast tissue squares (from left to right, respectively). image. (D) Visible light image of the chicken breast tissue square in panel C. (E) NIR fluorescence images superimposed on the visible light background of ICG-LNP capillaries placed 0.5 cm, 1.0 cm and 1.5 cm below chicken breast tissue squares. (F) Side view of tissue square thickness.
图3显示了ICG-LNP荧光强度以及荧光产量vs.游离ICG的荧光强度以及荧光产量。(A)脂质:ICG摩尔比对ICG荧光产量(每微摩尔ICG的荧光强度)的影响。制备具有不同脂质:ICG摩尔比的ICG-LNP，在标示的浓度条件下测量它们的每单位ICG的荧光强度。为了清楚起见，只提供八个ICG-LNP制剂中的三个，根据它们的脂质:ICG摩尔比标记它们的数据点：(●)250:1脂质:ICG摩尔比,(○)350:1脂质:ICG摩尔比,和(■)100:1脂质:ICG摩尔比以及(△)仅ICG。每个荧光强度数据点是八次重复测量的平均值±SD。(B)脂质:ICG摩尔比对荧光产量的影响。图A中的数据用于计算斜率(每微摩尔ICG的荧光强度)并且在不同摩尔比条件下相对于脂质:ICG摩尔比绘图：(○)仅游离ICG和(●)ICG-脂质纳米颗粒(ICG-LNP)。每个数据点是六次重复测量的平均值±SD。Figure 3 shows the fluorescence intensity and fluorescence yield of ICG-LNP vs. the fluorescence intensity and fluorescence yield of free ICG. (A) Effect of lipid:ICG molar ratio on ICG fluorescence yield (fluorescence intensity per micromole of ICG). ICG-LNPs with different lipid:ICG molar ratios were prepared, and their fluorescence intensity per unit ICG was measured at the indicated concentrations. For clarity, only three of the eight ICG-LNP preparations are presented, with their data points labeled according to their lipid:ICG molar ratio: (●) 250:1 lipid:ICG molar ratio, (○) 350: 1 lipid:ICG molar ratio, and (■) 100:1 lipid:ICG molar ratio and (△) ICG only. Each fluorescence intensity data point is the mean ± SD of eight replicate measurements. (B) Effect of lipid:ICG molar ratio on fluorescence yield. The data in panel A were used to calculate the slope (fluorescence intensity per micromole of ICG) and plotted against the lipid:ICG molar ratio under different molar ratio conditions: (○) free ICG only and (●) ICG-lipid nanoparticles Particles (ICG-LNP). Each data point is the mean±SD of six repeated measurements.
图4是肿瘤的检测，显示了分离的肿瘤的切片中ICG-LNP的荧光强度，所述肿瘤由执业病理学家表征为恶性纤维肉瘤(顶部)和鳞状细胞癌(底部)。Figure 4 is an examination of tumors showing the fluorescence intensity of ICG-LNP in sections of isolated tumors characterized by a practicing pathologist as malignant fibrosarcoma (top) and squamous cell carcinoma (bottom).
图5显示了来自两只不同的小鼠左腿下方皮肤处的图像。使用ICG-LNP检测到两只链脲佐菌素(STZ)1型糖尿病小鼠的皮肤中的炎症、淋巴管新生以及三级淋巴器官。Figure 5 shows images from the skin under the left leg of two different mice. Inflammation, lymphangiogenesis, and tertiary lymphoid organs were detected in the skin of two streptozotocin (STZ) type 1 diabetic mice using ICG-LNP.
图6显示了使用ICG-LNP在小鼠右腿中检测到带有血管周围单核细胞(淋巴细胞和组织细胞/巨噬细胞)累积的淋巴异常。该图像中小鼠的皮肤被除去。Figure 6 shows lymphoid abnormalities with accumulation of perivascular monocytes (lymphocytes and histiocytes/macrophages) detected in the right leg of mice using ICG-LNP. The skin of the mouse has been removed in this image.
图7显示了使用ICG-LNP检测到小鼠的左膝后窝淋巴结的左淋巴输入管中的淋巴管新生、淋巴管扩张以及三级淋巴器官。这些图像中的每一个中小鼠的皮肤已被除去。Figure 7 shows the detection of lymphangiogenesis, lymphangioectasia, and tertiary lymphoid organs in the left lymphatic afferent of the left posterior genuicular lymph nodes of mice using ICG-LNP. The skin of the mouse has been removed in each of these images.
图8显示了使用ICG-LNP检测到淋巴管中的淋巴管示踪和淋巴管扩张，在两只不同的小鼠中，所述淋巴管将左髂骨下(腹股沟)的淋巴结连接至左腋淋巴结。Figure 8 shows the use of ICG-LNP to detect lymphatic tracing and lymphatic dilatation in the lymphatic vessels connecting the left subiliac (inguinal) lymph nodes to the left axilla in two different mice lymph nodes.
图9显示了使用ICG-LNP检测到小鼠中的髂骨肌淋巴结、乳糜池以及胸淋巴导管的高分辨图像。Figure 9 shows high-resolution images of iliac muscle lymph nodes, chyle cisterns, and thoracic lymphatic ducts detected in mice using ICG-LNP.
图10显示了皮下足部注射之后淋巴结中的ICG-LNP的NIR荧光与游离ICG的NIR荧光比较。(A,B)腋窝淋巴结，(C,D)髂骨肌&胃淋巴结，(E,F,G,H)膝后窝&坐骨淋巴结。Figure 10 shows the NIR fluorescence of ICG-LNP in lymph nodes after subcutaneous foot injection compared to that of free ICG. (A,B) Axillary lymph nodes, (C,D) Iliac crest & gastric lymph nodes, (E,F,G,H) Posterior genital & ischial lymph nodes.
图11显示了足部皮下给药ICG-LNP的小鼠腋窝淋巴结的组织切片的NIR荧光显微图像。间断的荧光表明ICG-LNP颗粒的体内稳定性以及从注射位点通过淋巴系统沿着广泛路径进入淋巴结和淋巴组织的内部。一些ICG-LNP是细胞内的并且与巨噬细胞和树突细胞相关。Figure 11 shows NIR fluorescence microscopic images of tissue sections of axillary lymph nodes of mice administered subcutaneously with ICG-LNP on the foot. Intermittent fluorescence indicates the in vivo stability of ICG-LNP particles and the extensive pathway from the injection site through the lymphatic system into the interior of lymph nodes and lymphoid tissues. Some ICG-LNPs are intracellular and associated with macrophages and dendritic cells.
图12显示了ICG-LNP的储存稳定性。ICG-LNP和游离ICG在4℃下储存于暗处持续多达313天，并且在标记的时间点分析ICG荧光：(△)游离ICG和(●)ICG-脂质纳米颗粒(ICG-LNP)。基于指数衰减模型分析时间过程数据，并且列出半衰期t1/2(天数)值和速度常数k(天数的倒数)值。虚线表示检测限。每个数据点是三次至八次重复测量的平均值±标准差(SD)。Figure 12 shows the storage stability of ICG-LNP. ICG-LNP and free ICG were stored at 4°C in the dark for up to 313 days, and ICG fluorescence was analyzed at marked time points: (Δ) free ICG and (●) ICG-lipid nanoparticles (ICG-LNP) . Time course data were analyzed based on an exponential decay model, and half-life t1/2 (days) values and rate constant k (reciprocal of days) values are listed. Dashed lines indicate detection limits. Each data point is the mean ± standard deviation (SD) of three to eight replicate measurements.
图13显示了ICG-LNP减少了由于暴露于光线引起的ICG荧光淬灭。ICG-LNP和游离ICG暴露于顶部荧光光线持续12小时。在6小时和12小时记录ICG荧光。黑色柱代表ICG-LNP数据，灰色柱数据表示仅ICG。每个数据点是八次重复测量的平均值±SD。Figure 13 shows that ICG-LNP reduces ICG fluorescence quenching due to light exposure. ICG-LNP and free ICG were exposed to overhead fluorescent light for 12 hours. ICG fluorescence was recorded at 6 and 12 hours. Black bars represent ICG-LNP data, gray bar data represent ICG only. Each data point is the mean±SD of eight repeated measurements.
图14显示了ICG浓度依赖性LNP聚集以及表观尺寸增加。图A：ICG浓度对LNP的90°光散射强度的影响。在固定液量中，使用不同浓度的ICG孵育固定浓度的LNP。在第20分钟，稀释混合物以停止反应并且用荧光光度计测量90°光散射强度。空心圆符号表示只有空脂质的纳米颗粒，实心圆符号表示ICG脂质纳米颗粒(ICG-LNP)，并且空心三角形符号表示仅游离ICG。每个数据点是十次重复测量的平均值±SD。图B：由与入射光(hv)路径呈90°的光电倍增管(PMT，黑色水平柱)检测到的光散射效率随LNP聚集和粒度直径增加而发生变化的示意图。图C：通过LNP光子相关光谱分析ICG浓度对粒度的影响。在收集了图A的数据之后，进行粒度分析。空心圆符号表示只有空脂质的LNP，实心圆符号表示ICG脂质纳米颗粒(ICG-LNP)。每个数据点是数据收集八分钟之后由数字自动关联计算得到的平均值±SD。Figure 14 shows ICG concentration dependent LNP aggregation and apparent size increase. Panel A: The effect of ICG concentration on the 90° light scattering intensity of LNPs. A fixed concentration of LNP was incubated with different concentrations of ICG in a fixed volume. At 20 minutes, the mixture was diluted to stop the reaction and the 90° light scattering intensity was measured with a fluorometer. Open circle symbols represent nanoparticles with empty lipid only, solid circle symbols represent ICG lipid nanoparticles (ICG-LNP), and open triangle symbols represent free ICG only. Each data point is the mean±SD of ten repeated measurements. Panel B: Schematic representation of light scattering efficiency detected by a photomultiplier tube (PMT, black horizontal bars) at 90° to the incident light (hv) path as a function of LNP aggregation and particle size diameter. Panel C: Analysis of the effect of ICG concentration on particle size by LNP photon correlation spectroscopy. After the data for panel A was collected, particle size analysis was performed. Open circle symbols represent LNPs with only empty lipids, and closed circle symbols represent ICG lipid nanoparticles (ICG-LNP). Each data point is the mean ± SD calculated by numerical autocorrelation eight minutes after data collection.
图15显示了ICG脂质纳米颗粒(ICG-LNP)的荧光强度增加。在固定液量中，用不同浓度的ICG孵育固定浓度的LNP。在第20分钟，用缓冲液稀释混合物20倍以停止反应并用荧光光度计测量荧光强度，如实施例所描述的那样。空心三角形符号表示仅有游离的ICG，实心圆符号表示ICG脂质纳米颗粒(ICG-LNP)。每个数据点是八次重复测量的平均值±SD。Figure 15 shows the increase in fluorescence intensity of ICG lipid nanoparticles (ICG-LNP). A fixed concentration of LNP was incubated with different concentrations of ICG in a fixed volume. At 20 minutes, the reaction was stopped by diluting the mixture 20-fold with buffer and the fluorescence intensity was measured with a fluorophotometer, as described in the Examples. Open triangle symbols represent free ICG only, and solid circle symbols represent ICG lipid nanoparticles (ICG-LNP). Each data point is the mean±SD of eight repeated measurements.
图16显示了皮下注射之后小鼠体内的ICG脂质纳米颗粒与游离ICG NIR图像表现的比较。图A：皮下注射ICG脂质纳米颗粒(ICG-LNP，左足)或游离ICG(右足)并且在第六分钟收集NIR荧光图像。荧光图像重叠于可见光图像上，以用于解剖学表示。虚线圆表示局部膝后窝结。以仰卧位观察小鼠。在仅用游离ICG(图B)或ICG-LNP(图C)处理六分钟的另一组小鼠中，除去皮肤并进行进一步分析。图B：可见光图像是收集的在小鼠右足用游离ICG处理的小鼠的右腿图像。对应的荧光图像重叠于其上。双箭头(>>)表示游离ICG看起来扩散在整个肌肉组织中和膝后窝淋巴结(虚线圆)中的隐静脉。以仰卧位观察小鼠。图C：可见光图像是收集的在小鼠右足用ICG-LNP处理小鼠的右腿图像。将对应的荧光图像重叠于其上。虚线圆表示膝后窝淋巴结并且粗体箭头表示腹部骨盆和生殖器/局部淋巴结。以仰卧位观察小鼠。Figure 16 shows a comparison of ICG lipid nanoparticles and free ICG NIR image appearance in mice following subcutaneous injection. Panel A: ICG lipid nanoparticles (ICG-LNP, left foot) or free ICG (right foot) were injected subcutaneously and NIR fluorescence images were collected at 6 minutes. Fluorescence images are superimposed on visible light images for anatomical representation. Dashed circles indicate local posterior genital nodules. Observe the mice in the supine position. In another group of mice treated with free ICG (Panel B) or ICG-LNP (Panel C) for six minutes only, the skin was removed and further analyzed. Panel B: Visible light images were collected from the right leg of a mouse treated with free ICG on the right paw of the mouse. The corresponding fluorescence image is superimposed on it. Double arrows (>>) indicate the saphenous vein where free ICG appears to spread throughout the musculature and in the genital lymph nodes (dashed circle). Observe the mice in the supine position. Panel C: Visible light images were collected from the right leg of a mouse treated with ICG-LNP on the mouse's right paw. Overlay the corresponding fluorescence image on top of it. Dashed circles indicate genital lymph nodes and bold arrows indicate abdominal pelvic and genital/regional lymph nodes. Observe the mice in the supine position.
图17显示了在小鼠足部皮下给药后通过皮肤检测到的在第二引流淋巴结(坐骨淋巴结，从膝后窝坐骨淋巴结开始～2cm)中游离ICG和ICG-LNP的荧光强度的药物动力学。平均值±标准误(SEM)。图17显示了采用用于使注射位点的第二下游淋巴结(坐骨淋巴结)可视化的ICG-LNP实现了荧光强度的提高。使用ICG-LNP实现的荧光强度是游离ICG实现的荧光强度的两倍至三倍。仅采用ICG-LNP而不使用游离ICG还显示出清楚的分布和清除特特征。Figure 17 shows the pharmacokinetics of the fluorescence intensity of free ICG and ICG-LNP detected through the skin in the second draining lymph node (sciatic lymph node, ~2 cm from the ischial lymph node in the patella) after subcutaneous administration in the mouse foot study. Mean ± standard error (SEM). Figure 17 shows the increase in fluorescence intensity achieved with ICG-LNP for visualization of the second downstream lymph node (sciatic lymph node) from the injection site. The fluorescence intensity achieved with ICG-LNP was two to three times that of free ICG. ICG-LNP alone without free ICG also showed a clear distribution and clearance profile.
图18显示了在尾静脉内注射ICG-LNP之后通过皮肤检测到的肝脏药物动力学。当在小鼠尾静脉内IV注射ICG-LNP时，ICG-LNP容易从肝脏中清除，且终末消除半衰期为1.2小时。因此，在大约8小时(约7个半衰期)内ICG-LNP完全从肝脏中清除。这与传统脂质体的表现完全不同，传统脂质体由于Kupffer巨噬细胞的摄取而残留在肝脏中(Daemen T 1997Hepatology 26(2):416-423)。ICG-LNP从肝脏中通过胆管清除进入肠道。因此，ICG-LNP可用于评估肝-胆功能。Figure 18 shows hepatic pharmacokinetics detected by skin following tail vein injection of ICG-LNP. When ICG-LNP was injected IV in the tail vein of mice, ICG-LNP was easily cleared from the liver with a terminal elimination half-life of 1.2 hours. Thus, ICG-LNP is completely cleared from the liver within approximately 8 hours (approximately 7 half-lives). This is completely different from the behavior of traditional liposomes, which remain in the liver due to uptake by Kupffer macrophages (Daemen T 1997 Hepatology 26(2):416-423). ICG-LNP is cleared from the liver through the bile ducts into the intestine. Therefore, ICG-LNP can be used to assess hepato-biliary function.
图19显示了在正常小鼠、糖尿病小鼠或带有实体肿瘤的小鼠的足部皮下注射游离ICG和ICG-LNP之后，通过淋巴结(膝后窝和坐骨)的皮肤的体内荧光强度。图19显示了ICG-LNP在正常小鼠和患病(糖尿病或者带有肿瘤)小鼠中通过足部皮下注射位点检测膝后窝和坐骨淋巴结(分别检测第一和第二引流淋巴结)的能力。如上图1和表2、表3所示，ICG-LNP可用于评估这些疾病模型中的淋巴功能。在正常小鼠中，由ICG-LNP获得的比游离ICG更亮的图像表明获得增强的荧光强度和更高的分辨率是可能的。Figure 19 shows the in vivo fluorescence intensity through the skin of lymph nodes (posterior genital fossa and ischium) after subcutaneous injection of free ICG and ICG-LNP in the feet of normal mice, diabetic mice or mice with solid tumors. Figure 19 shows the detection of ICG-LNP in normal mice and diseased (diabetic or with tumor) mice through the subcutaneous injection site of the foot to detect the posterior knee fossa and ischial lymph nodes (detection of the first and second draining lymph nodes respectively) ability. As shown in Figure 1 and Tables 2 and 3 above, ICG-LNP can be used to assess lymphatic function in these disease models. In normal mice, brighter images obtained by ICG-LNP than free ICG indicated that it was possible to obtain enhanced fluorescence intensity and higher resolution.
图20显示了通过足部皮下给药ICG-LNP的小鼠的膝后窝淋巴结附近的皮肤检测淋巴管异常。图20显示了ICG-LNP通过皮肤检测淋巴管异常的能力。左图显示了小鼠右膝后窝淋巴结上方移动的异常淋巴管。在右图的正常小鼠中，没有检测到这种淋巴管。这些观察结果在将皮肤去除之后使皮肤下方淋巴管成像而得到证实。Figure 20 shows abnormality of lymphatic vessels detected in the skin near the posterior genu lymph nodes of mice subcutaneously administered ICG-LNP via the foot. Figure 20 shows the ability of ICG-LNP to detect lymphatic abnormalities through the skin. The image on the left shows abnormal lymphatic vessels moving over the right posterior patella lymph node in a mouse. In the normal mouse on the right, no such lymphatic vessels were detected. These observations were confirmed by imaging subcutaneous lymphatic vessels after the skin had been removed.
通过引用的并入incorporation by reference
本发明说明书中提到的所有公开出版物、专利和专利申请在此通过引用并入本文，其程度与具体且单独地说明每个单独的公开出版物、专利或专利申请通过引用并入本文相同。All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. .
具体实施方式detailed description
下文结合实例应用来描述本发明的几个方面，用于举例说明。应当理解的是，以下通过列举多种具体细节、相互关系以及方法以提供对本发明的全面理解。然而，本领域技术人员易于意识到，本发明可不采用具体细节中的一种或多种来实施或者可采用其他方法来实施。本发明不限于举例说明的操作顺序或事件顺序，一些操作可以不同的顺序发生和/或与其他操作或事件同时发生。Several aspects of the invention are described below with reference to example applications, for purposes of illustration. It should be understood that the following set forth numerous specific details, interrelationships, and methodologies in order to provide a thorough understanding of the invention. One skilled in the art will readily appreciate, however, that the invention may be practiced without one or more of the specific details or may be practiced otherwise. The invention is not limited to the illustrated order of operations or events, and some operations may occur in different orders and/or concurrently with other operations or events.
而且，不要求将所有举例说明的操作或事件都用于实施根据本发明的方法。Moreover, not all illustrated operations or events are required to implement methods in accordance with the present invention.
术语“约”或“大约”意指由本领域技术人员确定的特定数值在可接受的误差范围内，所述可接受的误差范围部分取决于如何测定或确定所述数值，即，测量系统的限制。例如，根据本领域的实践，“约”可指在1或大于1的标准偏差范围内。可选地，“约”可指给定值的高达20％的范围，优选地，给定值的高达10％的范围，更优选地，给定值的高达5％的范围，以及更加优选地，给定值的高达1％的范围。可选地，尤其针对生物系统或过程，术语“约”可指数值的某数量级范围内，优选地，数值的5倍范围内，更优选地，数值的2倍范围内。在本申请和权利要求中描述具体数值时，除非另有说明，应当假设术语“约”是指该具体数值在可接受的误差范围内。The term "about" or "approximately" means that the particular value as determined by one skilled in the art is within an acceptable error range which depends in part on how the value was measured or determined, i.e., the limitations of the measurement system . For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" may refer to a range of up to 20% of a given value, preferably up to 10% of a given value, more preferably up to 5% of a given value, and even more preferably , a range of up to 1% of the given value. Alternatively, particularly for biological systems or processes, the term "about" may refer to within a certain order of magnitude of a value, preferably within 5 times the value, more preferably within 2 times the value. Where specific values are described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean that the specific value is within an acceptable error range.
除非另有说明，本文使用的所有技术和科学术语总体上具有与本领域技术人员通常理解的含义相同的含义。通常，本文使用的命名法和在细胞培养、细胞遗传学、有机化学和核酸化学以及杂交中的实验步骤是本领域所熟知且常用的。标准技术用于核酸和肽合成。技术和步骤通常根据本领域的常规方法和各种常用参考文献来实施，这些常规方法和常用参考文献在全文中提供。本文使用的命名法和下文描述的分析化学和有机合成的实验步骤是本领域所熟知的并且是本领域常用的。标准技术或其改良用于化学合成和化学分析。Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, cytogenetics, organic and nucleic acid chemistry, and hybridization are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods in the art and various common references, which are provided throughout. The nomenclature used herein and the analytical chemistry and organic synthesis procedures described below are those well known and commonly used in the art. Standard techniques or modifications thereof were used for chemical syntheses and chemical analyses.
I.组合物I. Composition
本发明涉及生物医学成像的组合物以及应用，更加具体而言，本发明涉及独特的制备方法、组合物和直径为纳米尺寸的用于近红外荧光(NIRF)医学成像的红外荧光发光颗粒，所述成像包括血液成像、组织成像以及淋巴脉管系统和淋巴结(LN)成像。The present invention relates to the composition and application of biomedical imaging, more specifically, the present invention relates to a unique preparation method, composition and infrared fluorescent particles with diameters of nanometer size for near-infrared fluorescence (NIRF) medical imaging, so Such imaging includes blood imaging, tissue imaging, and lymphatic vasculature and lymph node (LN) imaging.
一方面，本发明提供一种组合物，所述组合物包含：(i)含有脂质膜和水性核的脂质纳米颗粒(例如，球体)；和(ii)多个吲哚菁绿(ICG)，其中，多个ICG中的一个或多个嵌入所述纳米颗粒的脂质膜。In one aspect, the invention provides a composition comprising: (i) lipid nanoparticles (e.g., spheres) comprising a lipid membrane and an aqueous core; and (ii) a plurality of indocyanine green (ICG ), wherein one or more of the plurality of ICGs is embedded in the lipid membrane of the nanoparticle.
A.脂质纳米颗粒A. Lipid nanoparticles
总体而言，本文提供的组合物包含纳米颗粒。In general, the compositions provided herein comprise nanoparticles.
本发明的纳米颗粒的尺寸可为约20nm至20μm，例如，约20nm至5μm，约80nm至2μm，约80nm至1μm。因此，虽然本文中称为“纳米颗粒”，但是微米尺度的颗粒也包括在本文的范围内。对于静脉内或动脉内注射给药而言，颗粒尺寸优选为约80nm至100nm。对于其他给药途径而言，例如，经鼻给药，可使用较大的颗粒。在一些实施方式中，使用尺寸为约20nm至300nm，例如，约20nm至100nm，20nm至50nm的颗粒。每种可能性代表本发明的单独的实施方式。The size of the nanoparticles of the present invention may be about 20 nm to 20 μm, eg, about 20 nm to 5 μm, about 80 nm to 2 μm, about 80 nm to 1 μm. Thus, although referred to herein as "nanoparticles," micron-scale particles are also included within the scope of this document. For intravenous or intraarterial injection administration, the particle size is preferably about 80 nm to 100 nm. For other routes of administration, eg, nasal administration, larger particles may be used. In some embodiments, particles with a size of about 20 nm to 300 nm, eg, about 20 nm to 100 nm, 20 nm to 50 nm, are used. Each possibility represents a separate embodiment of the invention.
在一些实施方式中，颗粒的粒度不受特定限制，条件是当所述颗粒用作对比剂时，尤其是用于淋巴结的对比剂时，将所述颗粒的流体动力学平均粒度设定为1,000nm或更小可提高所述颗粒被淋巴管或组织摄取的容易性(组织渗透性)及其在淋巴结或组织中的保持力。In some embodiments, the particle size of the particles is not particularly limited, provided that when the particles are used as a contrast agent, especially for lymph nodes, the hydrodynamic average particle size of the particles is set to 1,000 nm or less improves the ease of uptake of the particles into lymphatic vessels or tissues (tissue permeability) and their retention in lymph nodes or tissues.
当粒度为1,000nm或更小时，相对于在生物体的正常位点的积累量，更大量的颗粒可通过提高的渗透性和滞留效应(EPR)积累在生物体的肿瘤位点。肿瘤位点可通过检测具有各种不同的成像形态(例如，荧光和光声学)的积累的颗粒而进行特异性成像。此外，当粒度超过1,000nm时，无法预期在诸如淋巴管之类的组织中的有效摄取。因此，平均粒度优选地设定为10nm或更大以及1,000nm或更小。平均粒度更加优选地为20nm或更大以及500nm或更小，尤为优选地为20nm或更大以及200nm或更小，特别优选地为20nm至100nm。这是因为当颗粒的粒度为200nm或更小时，颗粒很难被血液中的巨噬细胞摄取，因此，可改善颗粒在血液中的保持力。When the particle size is 1,000 nm or less, a larger amount of particles can accumulate at the tumor site of the organism through enhanced permeability and retention effect (EPR) relative to the accumulated amount at the normal site of the organism. Tumor sites can be specifically imaged by detecting accumulated particles with various imaging modalities (eg, fluorescence and photoacoustics). Furthermore, when the particle size exceeds 1,000 nm, efficient uptake in tissues such as lymphatic vessels cannot be expected. Therefore, the average particle size is preferably set to 10 nm or more and 1,000 nm or less. The average particle size is more preferably 20 nm or more and 500 nm or less, particularly preferably 20 nm or more and 200 nm or less, particularly preferably 20 nm to 100 nm. This is because when the particles have a particle size of 200 nm or less, the particles are hardly taken up by macrophages in the blood, and therefore, the retention of the particles in the blood can be improved.
粒度可通过采用电子显微镜观察测量或可通过基于动态光散射法的粒度测量方法进行测量。当基于动态光散射法测量粒度时，采用动态光散射分析仪(Particle Sizing Systems,Port Richey,FL生产的PSS-NICOMP 380ZLS仪器)由动态光散射(DLS)法测量流体动力学直径。The particle size can be measured by observation using an electron microscope or can be measured by a particle size measurement method based on a dynamic light scattering method. When the particle size is measured based on the dynamic light scattering method, the hydrodynamic diameter is measured by the dynamic light scattering (DLS) method using a dynamic light scattering analyzer (PSS-NICOMP 380ZLS instrument manufactured by Particle Sizing Systems, Port Richey, FL).
本文使用的术语“约”涉及诸如量或尺寸之类的可测量的数值时，意在包括特定值的+/-10％的变量，更加优选地+/-5％的变量，甚至更加优选地+/-1％的变量，以及尤为优选地+/-0.1％的变量，这些变量适于实现所期望的目的。As used herein, the term "about" when referring to a measurable numerical value such as an amount or size is intended to include a variation of +/- 10% of the specified value, more preferably a variation of +/- 5%, even more preferably A variation of +/-1%, and especially preferably a variation of +/-0.1%, is suitable to achieve the desired purpose.
本文使用的纳米颗粒的“尺寸”是指纳米颗粒的最长维度(宽度、长度或直径)在指定的范围内。通常，含有纳米颗粒的制剂中的平均粒度在指定范围内。所述纳米颗粒可具有均一的形状，例如，球形或细长形，或可具有多种形状。优选地，本文的纳米颗粒是球形。As used herein, the "size" of a nanoparticle means that the longest dimension (width, length or diameter) of the nanoparticle is within the specified range. Typically, the average particle size in formulations containing nanoparticles is within the specified range. The nanoparticles may have a uniform shape, eg, spherical or elongated, or may have a variety of shapes. Preferably, the nanoparticles herein are spherical.
在一些实施方式中，纳米颗粒的平均尺寸为50nm至100nm，优选地为50nm至80nm。In some embodiments, the nanoparticles have an average size of 50 nm to 100 nm, preferably 50 nm to 80 nm.
总体而言，本文提供的组合物(例如，药物组合物)中的纳米颗粒具有约50±5nm,约55±5nm,约60±5nm,约65±5nm,约70±5nm,约75±5nm,约80±5nm,约85±5nm,约90±5nm,约95±5nm,或约100±5nm的均一性。在一些实施方式中，LNP具有±1nm,±2nm,±3nm,±4nm,或±5nm的均一性。Generally, the nanoparticles in the compositions provided herein (e.g., pharmaceutical compositions) have a particle size of about 50±5 nm, about 55±5 nm, about 60±5 nm, about 65±5 nm, about 70±5 nm, about 75±5 nm , about 80±5nm, about 85±5nm, about 90±5nm, about 95±5nm, or about 100±5nm uniformity. In some embodiments, the LNP has a uniformity of ±1 nm, ±2 nm, ±3 nm, ±4 nm, or ±5 nm.
本发明的纳米颗粒可带有正电荷或负电荷。本文使用的纳米颗粒的“电荷”是指它们的表面电荷，称为ζ电位。对于静脉内给药或动脉内给药而言，带有负电荷的颗粒目前是优选的。带负电荷的颗粒的表面电荷(ζ电位)的范围可为约-20mV至-55mV。The nanoparticles of the invention can be positively or negatively charged. As used herein, the "charge" of nanoparticles refers to their surface charge, known as the zeta potential. For intravenous or intraarterial administration, negatively charged particles are presently preferred. The surface charge (zeta potential) of negatively charged particles may range from about -20 mV to -55 mV.
粒度和ζ电位的测量可通过本领域已知的方法进行，例如，使用商售仪器(例如，Zetasizer NanoZS(Malvern,UK))通过动态光散射(DLS)进行粒度和ζ电位的测量。Measurements of particle size and zeta potential can be performed by methods known in the art, for example, by dynamic light scattering (DLS) using a commercially available instrument (eg, Zetasizer NanoZS (Malvern, UK)).
在一些实施方式中，纳米颗粒带有约-5mV至-100mV的表面负电荷。In some embodiments, the nanoparticles have a negative surface charge of about -5 mV to -100 mV.
在一些实施方式中，本发明的纳米颗粒是脂质体。In some embodiments, nanoparticles of the invention are liposomes.
本发明中使用的脂质颗粒可被制备成包括脂质体成型脂质和磷脂以及膜活性固醇(例如，胆固醇)。脂质体可包括其他不是脂质体成型脂质的脂质和磷脂。Lipid particles used in the invention can be prepared to include liposome-forming lipids and phospholipids as well as membrane active sterols (eg, cholesterol). Liposomes can include other lipids and phospholipids that are not liposome-forming lipids.
磷脂可选自例如：卵磷脂(例如，鸡蛋或大豆卵磷脂)，磷脂酰胆碱(例如，鸡蛋磷脂酰胆碱)，氢化的磷脂酰胆碱，溶血磷脂酰胆碱，二棕榈酰基磷脂酰胆碱，二硬脂酰基磷脂酰胆碱，二肉豆蔻酰基磷脂酰胆碱，双月桂酰基磷脂酰胆碱，甘油磷脂(例如，磷脂酰甘油，磷脂酰丝氨酸，磷脂酰乙醇胺，溶血磷脂酰乙醇胺，磷脂酰肌醇，磷脂酰肌醇磷酸，磷脂酰肌醇双磷酸和磷脂酰肌醇三磷酸)，鞘磷脂，心磷脂，磷脂酸，缩醛磷脂，或者它们的混合物。每种可能性代表本发明的单独的实施方式。Phospholipids may be selected from, for example: lecithin (e.g. egg or soybean lecithin), phosphatidylcholine (e.g. egg phosphatidylcholine), hydrogenated phosphatidylcholine, lysophosphatidylcholine, dipalmitoylphosphatidylcholine Choline, Distearoylphosphatidylcholine, Dimyristoylphosphatidylcholine, Dilauroylphosphatidylcholine, Glycerophospholipids (eg, Phosphatidylglycerol, Phosphatidylserine, Phosphatidylethanolamine, Lysophosphatidylethanolamine , phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate), sphingomyelin, cardiolipin, phosphatidic acid, plasmalogen, or mixtures thereof. Each possibility represents a separate embodiment of the invention.
可使用的其他脂质的实例包括糖脂(例如，甘油糖脂，例如，半乳糖脂和硫代脂质，鞘糖脂，例如，脑苷脂，葡萄糖脑苷脂和半乳糖脑苷脂，以及糖基磷脂酰肌醇)，鞘磷脂(例如，神经酰胺磷酸胆碱，神经酰胺磷酰基乙醇胺和神经酰胺磷酰基甘油)，或者它们的混合物。每种可能性表示本发明的单独的实施方式。Examples of other lipids that can be used include glycolipids (e.g., glyceroglycolipids, e.g., galactolipids and thiolipids, glycosphingolipids, e.g., cerebrosides, glucocerebrosides, and galactocerebrosides, and glycosylphosphatidylinositol), sphingomyelin (eg, ceramide phosphorylcholine, ceramide phosphoryl ethanolamine, and ceramide phosphoryl glycerol), or mixtures thereof. Each possibility represents a separate embodiment of the invention.
带负电荷或带正电荷的脂质纳米颗粒可例如通过使用阴离子或阳离子磷脂或脂质获得。这些阴离子/阳离子磷脂或脂质通常具有亲脂基团，例如，固醇，酰基或二酰基链并且其中，脂质具有总体净负电荷/正电荷。Negatively or positively charged lipid nanoparticles can be obtained, for example, by using anionic or cationic phospholipids or lipids. These anionic/cationic phospholipids or lipids typically have lipophilic groups, eg, sterols, acyl or diacyl chains and where the lipids have an overall net negative/positive charge.
上述脂质和磷脂可购买获得或根据本领域公开的方法制备。The aforementioned lipids and phospholipids are commercially available or prepared according to methods disclosed in the art.
脂质颗粒可通过本领域已知的方法制备，参见例如Scholar等人,2012,International Journal of Pharmaceutical Studies and Research,3(2):14-20；Akbarzadeh等人,2013,Nanoscale Research Letters,8:102中公开的方法。示例性的方法在下文中描述。通过小孔膜(例如聚碳酸酯膜)挤出脂质体是将尺寸降低至相对明确界定的尺寸分布的有效方法。通常，使悬浮液循环穿过膜几次(使用孔径递减的膜)，直至得到理想的脂质体尺寸分布。连续通过孔较小的膜挤出脂质颗粒能够逐步将脂质体尺寸降低至理想尺寸。经过尺寸降低处理的脂质颗粒悬浮液可易于通过颗粒识别尺寸为例如约0.2μm的灭菌膜进行杀菌，例如常规0.22μm厚的膜过滤器。如果需要的话，脂质体悬浮液可在存在合适的储存用冷冻保护剂的条件下被冻干并在使用前通过简单水化而复溶。Lipid particles can be prepared by methods known in the art, see for example Scholar et al., 2012, International Journal of Pharmaceutical Studies and Research, 3(2):14-20; Akbarzadeh et al., 2013, Nanoscale Research Letters, 8: The method disclosed in 102. Exemplary methods are described below. Extrusion of liposomes through a small pore membrane, such as a polycarbonate membrane, is an efficient method for size reduction to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane several times (using membranes of decreasing pore size) until the desired liposome size distribution is obtained. Continuous extrusion of lipid particles through a membrane with smaller pores can gradually reduce the liposome size to the desired size. The size-reduced lipid particle suspension can be readily sterilized by passage through a sterilization membrane having a particle recognition size of, eg, about 0.2 μm, such as a conventional 0.22 μm thick membrane filter. If desired, liposomal suspensions can be lyophilized in the presence of a suitable cryoprotectant for storage and reconstituted by brief hydration before use.
总体而言，如本文更加详细地描述的，本发明的脂质纳米颗粒通过将诸如ICG之类的染料与脂质分子混合，随后产生颗粒来制备。In general, as described in more detail herein, lipid nanoparticles of the invention are prepared by mixing a dye, such as ICG, with lipid molecules, followed by particle generation.
在一些实施方式中，形成纳米颗粒的脂质分子包括两种磷脂种类，1,2-二硬脂酰基-sn-丙三基-3-磷酸胆碱(DSPC)和1,2-二硬脂酰基-sn-丙三基-3-磷酸乙醇胺-N-甲氧基-聚乙二醇-2000(DSPEmPEG2000)。In some embodiments, the lipid molecules forming the nanoparticles include two phospholipid species, 1,2-distearoyl-sn-propanetriyl-3-phosphocholine (DSPC) and 1,2-distearyl Acyl-sn-glyceryl-3-phosphoethanolamine-N-methoxy-polyethylene glycol-2000 (DSPEmPEG2000).
B.荧光染料B. Fluorescent dyes
在一些实施方式中，本发明的纳米颗粒包含至少一种嵌入脂质中的近红外(NIR)荧光染料(或探针)。In some embodiments, nanoparticles of the invention comprise at least one near-infrared (NIR) fluorescent dye (or probe) embedded in lipids.
在一些实施方式中，结合是非共价结合。In some embodiments, the binding is non-covalent.
本文中的“近红外(NIR)荧光染料”或“近红外(NIR)荧光探针”是指适于成像应用的分子或实体，其能够在NIR光谱范围内吸收和发射光。具体而言，本文中的“近红外(NIR)荧光染料”或“近红外(NIR)荧光探针”是具有在NIR光谱范围内，优选地在约700nm至900nm的范围内的激发光和发射光的荧光实体。NIR辐射通常限定为具有约700nm至1400nm的波长。本发明的NIR荧光探针优选地是那些吸收和发射在约700nm至900nm的范围内的NIR光的NIR荧光探针，所述约700nm至900nm的范围被认为是生物“NIR窗口”。A "near-infrared (NIR) fluorescent dye" or "near-infrared (NIR) fluorescent probe" herein refers to a molecule or entity suitable for imaging applications that is capable of absorbing and emitting light in the NIR spectral range. Specifically, "near-infrared (NIR) fluorescent dyes" or "near-infrared (NIR) fluorescent probes" herein are those having excitation light and emission in the NIR spectral range, preferably in the range of about 700nm to 900nm. A fluorescent entity of light. NIR radiation is generally defined as having a wavelength of about 700 nm to 1400 nm. The NIR fluorescent probes of the present invention are preferably those that absorb and emit NIR light in the range of about 700 nm to 900 nm, which is considered the biological "NIR window".
合适的NIR荧光探针的实例包括染料，例如，花青染料，例如，吲哚菁绿(ICG)，Cy5，Cy5.5，Cy5.18，Cy7和Cy7.5；ALEXA染料,染料,ANGIOS TAMPTM染料,SENTIDYETM染料,XENOLIGHT DIRTM荧光染料,VIVOTRACKTMNIR荧光显像剂,KODAKX-SIGHTTM染料和共轭物,DYLIGHTTM染料。NIR量子点也可用作探针(NIR量子点的合成和功能化在例如Ma等人,2010,Analyst,135:1867-1877中描述)。每种可能性代表本发明的单独的实施方式。其他染料包括亚甲基蓝、ProSense和MMPSense。Figueiredo,J.L.,Alencar,H.,Weissleder,R.&Mahmood,U.Nearinfrared thoracoscopy of tumoral protease activity for improved detection ofperipheral lung cancer.Int.J.Cancer 118,2672–2677(2006),以及Nguyen,Q.T.,和Tsien,R.Y.(2013)Fluorescence-guided surgery with live molecularnavigation:A new cutting edge.Nat.Rev.13,653-662。Examples of suitable NIR fluorescent probes include dyes, e.g., cyanine dyes, e.g., Indocyanine Green (ICG), Cy5, Cy5.5, Cy5.18, Cy7 and Cy7.5; ALEXA dye, Dyes, ANGIOS TAMPTM dyes, SENTIDYETM dyes, XENOLIGHT DIRTM fluorescent dyes, VIVOTRACKTM NIR fluorescent imaging agents, KODAKX-SIGHTTM dyes and conjugates, DYLIGHTTM dyes. NIR quantum dots can also be used as probes (synthesis and functionalization of NIR quantum dots is described eg in Ma et al., 2010, Analyst, 135:1867-1877). Each possibility represents a separate embodiment of the invention. Other dyes include methylene blue, ProSense, and MMPSense. Figueiredo, JL, Alencar, H., Weissleder, R. & Mahmood, U. Nearinfrared thoracoscopy of tumoral protease activity for improved detection of peripheral lung cancer. Int. J. Cancer 118, 2672–2677 (2006), and Nguyen, QT, and Tsien, RY (2013) Fluorescence-guided surgery with live molecular navigation: A new cutting edge. Nat. Rev. 13, 653-662.
在一些实施方式中，荧光染料是亲脂性(Log P>2)荧光染料，分子量为300-1500g/mol，例如，吲哚菁绿，亚甲基蓝，ProSense和MMPSense。In some embodiments, the fluorescent dye is a lipophilic (Log P>2) fluorescent dye with a molecular weight of 300-1500 g/mol, eg, indocyanine green, methylene blue, ProSense and MMPSense.
下表列出了可嵌入本发明的脂质纳米颗粒中的其他NIR染料。任何发荧光的疏水小分子可嵌入本发明的脂质纳米颗粒中。The table below lists other NIR dyes that can be embedded in the lipid nanoparticles of the invention. Any small hydrophobic molecule that fluoresces can be embedded in the lipid nanoparticles of the invention.
近红外荧光染料列表。List of near-infrared fluorescent dyes.
用于本发明的纳米颗粒的NIR荧光探针的特定实施方式是吲哚菁绿(ICG)。A particular embodiment of the NIR fluorescent probe used in the nanoparticles of the invention is indocyanine green (ICG).
ICG是临床上用于NIRF成像的染料(具有820nm的发射波长)并且是唯一由美国食品和药品管理局(FDA)和欧洲药物管理局(EMA)批准的用于人体的NIRF分子。ICG被批准用于心输出量、肝脏功能和肝脏血流检测，以及眼部血管造影术。ICG存在着大量的标签外和以研究为目的的使用情况，包括液体填充的解剖学结构(例如，血液、脑脊液、淋巴或尿液)的可视化，或作为对比剂用于血管、肾脏或其它排泄途径的显影(1)。在水性环境中，ICG分子聚集并且ICG荧光强度容易衰减(降低整体荧光强度)(2-5)。在血液中，ICG与血浆蛋白结合，部分且暂时地改善了其荧光强度，但是最终仍然会与血浆蛋白解离进入水相环境中被降解。在体内，ICG荧光强度和持续时间可随血浆蛋白和脂蛋白浓度的波动以及个体之间的差异而发生变化。ICG is a clinically used dye for NIRF imaging (with an emission wavelength of 820 nm) and is the only NIRF molecule approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for use in humans. ICG is approved for cardiac output, liver function, and liver blood flow testing, as well as ocular angiography. Numerous off-label and research-oriented uses of ICG exist, including visualization of fluid-filled anatomical structures (eg, blood, cerebrospinal fluid, lymph, or urine), or as a contrast agent for vascular, renal, or other excretory Visualization of pathways (1). In an aqueous environment, ICG molecules aggregate and ICG fluorescence intensity tends to decay (reduce overall fluorescence intensity) (2-5). In blood, ICG binds to plasma proteins, partially and temporarily improving its fluorescence intensity, but eventually dissociates from plasma proteins and enters the aqueous environment for degradation. In vivo, ICG fluorescence intensity and duration can vary with fluctuations in plasma protein and lipoprotein concentrations and with interindividual variability.
本发明的纳米颗粒可包括高达约10％(w/w)的NIR荧光探针，例如高达约5％(w/w)的NIR荧光探针，高达约1％(w/w)的NIR荧光探针，高达约0.5％(w/w)的NIR荧光探针，约0.005％-5％(w/w)的NIR荧光探针。The nanoparticles of the invention may comprise up to about 10% (w/w) NIR fluorescent probes, for example up to about 5% (w/w) NIR fluorescent probes, up to about 1% (w/w) NIR fluorescent Probes, up to about 0.5% (w/w) NIR fluorescent probe, about 0.005%-5% (w/w) NIR fluorescent probe.
C.ICG-LNPC.ICG-LNP
另一方面，本发明提供ICG-LNP，其中，ICG中的一个或多个嵌入纳米颗粒的脂质中。In another aspect, the invention provides ICG-LNP, wherein one or more of the ICGs are embedded in the lipid of the nanoparticle.
脂质体包裹的ICG(即，ICG在脂质体的水性核内)(8-12)导致ICG和脂质之间发生弱相互作用。Liposome-encapsulated ICG (ie, ICG within the aqueous core of the liposome) (8-12) results in weak interactions between ICG and lipids.
为了克服这些限制，尝试将ICG加至疏水聚合物、血清白蛋白和脂质体中。不是所有脂质体和脂质纳米颗粒都具有类似结构，一些脂质体和脂质纳米颗粒可能由具有不同头部基团和脂肪酰基链的磷脂构成，而其他脂质体和脂质纳米颗粒可包括胆固醇或其他添加物，所有这些均可改变脂质体的生理化学性质(6)。脂质的极性头部基团影响颗粒表面电荷和水合程度，其影响体内调理素作用的水平和引起清除的补体结合的水平，并且脂质脂肪酰基尾部的饱和量和链长通过改变脂质双层的刚性、厚度和均匀性影响脂质相变温度(Tc)(6)。因此，每个变量可能不仅仅影响ICG和脂质之间的稳定性和相互作用，并且还影响颗粒的体内行为。To overcome these limitations, attempts have been made to incorporate ICG into hydrophobic polymers, serum albumin and liposomes. Not all liposomes and lipid nanoparticles have similar structures, and some liposomes and lipid nanoparticles may be composed of phospholipids with different head groups and fatty acyl chains, while others Cholesterol or other additives may be included, all of which can modify the physiochemical properties of the liposomes (6). The polar headgroups of lipids affect the particle surface charge and degree of hydration, which affect the level of opsonization in vivo and the level of complement fixation leading to clearance, and the saturation amount and chain length of lipid fatty acyl tails by changing lipid The rigidity, thickness and homogeneity of the bilayer affect the lipid phase transition temperature (Tc) (6). Therefore, each variable may not only affect the stability and interaction between ICG and lipids, but also affect the behavior of particles in vivo.
本发明提供具有很强(不可逆地结合)的ICG-脂质相互作用的新型ICG-LNP，所述ICG-脂质相互作用由本发明提供的将ICG嵌入脂质颗粒中而产生。制备脂质体包裹的ICG需要纯化/过滤步骤以除去游离(未包裹的)ICG，在本发明中不需要进行所述纯化/过滤步骤，因为在本发明中能够达到脂质中基本上完全100％的嵌入效率。The present invention provides novel ICG-LNPs with strong (irreversibly bound) ICG-lipid interactions resulting from the embedding of ICG into lipid particles provided by the present invention. Preparation of liposome-encapsulated ICG requires a purification/filtration step to remove free (unencapsulated) ICG, which is not required in the present invention since essentially complete 100 in lipids can be achieved in the present invention. % embedding efficiency.
ICG-LNP的一个重要特性是其减低或甚至消除与ICG的浓度依赖性自淬灭性质有关的缺陷。参见图3。因为ICG的吸收光谱和发射光谱高度重叠，所以ICG表现出浓度依赖性荧光淬灭(自淬灭)。因此，重要的是，不仅仅限定脂质-ICG的相互作用，而且还限定脂质中ICG分子的最佳密度，所述最佳密度表现出每个ICG分子的最大荧光强度和最小自淬灭。参见图3。与大部分红外染料的普遍荧光性质相反，ICG在浓度低至几微摩尔(μM)ICG时发生自淬灭。虽然已对脂质体和脂质-ICG混合物进行了很多研究，但是没有研究从整体上考虑自淬灭性质，该自淬灭性质导致荧光稳定性和强度发生改变，并且更加重要的是，实现本发明的改进(通过最小化自淬灭)。本发明提供简单的三步骤程序产生稳定的ICG脂质纳米颗粒产品，用于高分辨率的NIRF成像。KraftJC,Ho RJ(2014)Interactions of indocyanine green and lipid in enhancingnear-infrared fluorescence properties:the basis for near-infrared imaging in vivo.Biochemistry；Mar 4；53(8):1275-83，该参考文献的全部内容通过引用并入本文。An important property of ICG-LNP is that it reduces or even eliminates the defects associated with the concentration-dependent self-quenching properties of ICG. See Figure 3. Because the absorption and emission spectra of ICG highly overlap, ICG exhibits concentration-dependent fluorescence quenching (self-quenching). Therefore, it is important not only to define the lipid-ICG interaction, but also to define the optimal density of ICG molecules in the lipid that exhibits the maximum fluorescence intensity and minimum self-quenching per ICG molecule . See Figure 3. In contrast to the ubiquitous fluorescent properties of most infrared dyes, ICG self-quenches at concentrations as low as a few micromolar (μM) ICG. Although much research has been done on liposomes and lipid-ICG mixtures, none have considered the self-quenching properties as a whole, which lead to changes in fluorescence stability and intensity and, more importantly, to achieve Improvement of the present invention (by minimizing self-quenching). The present invention provides a simple three-step procedure to generate stable ICG lipid nanoparticle products for high-resolution NIRF imaging. KraftJC, Ho RJ (2014) Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo. Biochemistry; Mar 4; 53(8):1275-83, the entire content of this reference Incorporated herein by reference.
在一些实施方式中，至少约90％,91％,92％,93％,94％,95％,96％,97％,97.5％,98％,98.5％,99％,99.5％或99.9％的ICG嵌入脂质膜中。在一些实施方式中，偏差为+/-0.1％,+/-0.2％,+/-0.3％,+/-0.4％,+/-0.5％,+/-0.6％,+/-0.7％,+/-0.8％,+/-0.9％,+/-10.0％。在一些实施方式中，至少97.8+/-0.6％的ICG嵌入脂质膜中。In some embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% of ICG is embedded in lipid membranes. In some embodiments, the deviations are +/-0.1%, +/-0.2%, +/-0.3%, +/-0.4%, +/-0.5%, +/-0.6%, +/-0.7%, +/-0.8%, +/-0.9%, +/-10.0%. In some embodiments, at least 97.8 +/- 0.6% of the ICG is embedded in the lipid membrane.
在一些实施方式中，基本上或全部100％的ICG嵌入脂质膜中。In some embodiments, substantially or all 100% of the ICG is embedded in the lipid membrane.
在一些实施方式中，少于5％,4％,3％,2％,1％,0.9％,0.8％,0.6％,0.5％,0.4％,0.3％,0.2％,0.1％,0.01％或0.001％的ICG包裹在脂质纳米颗粒的水性核中。In some embodiments, less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01%, or 0.001% ICG is encapsulated in the aqueous core of lipid nanoparticles.
在一些实施方式中，没有可检测量的ICG包裹在脂质纳米颗粒的水性核中。In some embodiments, no detectable amount of ICG is encapsulated in the aqueous core of the lipid nanoparticle.
嵌入脂质膜或包裹在脂质纳米颗粒的水性核中的ICG的量或百分比使用本领域已知的方法测量，例如，Bilayer/water partitioning studies by equilibriumdialysis,Joguparthi V,Feng S,Anderson BD.Determination of intraliposomal pHand its effect on membrane partitioning and passive loading of a hydrophobiccamptothecin,DB-67.Int J Pharm.2008Mar 20；352(1-2):17-28.Epub 2007Oct12.PubMed PMID:18065174；PubMed Central PMCID:PMC2277365，该参考文献的全部内容通过引用并入本文。The amount or percentage of ICG embedded in the lipid membrane or encapsulated in the aqueous core of lipid nanoparticles is measured using methods known in the art, for example, Bilayer/water partitioning studies by equilibriumdialysis, Joguparthi V, Feng S, Anderson BD. Determination of intraliposomal pH and its effect on membrane partitioning and passive loading of a hydrophobiccamptothecin, DB-67.Int J Pharm.2008Mar 20;352(1-2):17-28.Epub 2007Oct12.PubMed PMID:18065174;PubMed Central PMCID73665 , the entire content of which reference is incorporated herein by reference.
在一些实施方式中，ICG与脂质的比例为约0.3至约0.2mol/mol，0.3至0.1mol/mol，0.3至0.09mol/mol，0.3至0.08mol/mol，0.3至0.07mol/mol，0.3至0.06mol/mol，0.3至0.05mol/mol，0.3至0.04mol/mol，0.3至0.03mol/mol，0.3至0.02mol/mol或0.3至0.001mol/mol。In some embodiments, the ratio of ICG to lipid is about 0.3 to about 0.2 mol/mol, 0.3 to 0.1 mol/mol, 0.3 to 0.09 mol/mol, 0.3 to 0.08 mol/mol, 0.3 to 0.07 mol/mol, 0.3 to 0.06 mol/mol, 0.3 to 0.05 mol/mol, 0.3 to 0.04 mol/mol, 0.3 to 0.03 mol/mol, 0.3 to 0.02 mol/mol or 0.3 to 0.001 mol/mol.
在一些实施方式中，在脂质嵌入组合物中使用的ICG的剂量是水性ICG(临床使用)剂量的十分之一，如在每个患者/程序中使用的ICG-LNP的量中所反映的。In some embodiments, the dose of ICG used in the lipid-embedded composition is one-tenth the dose of aqueous ICG (clinically used), as reflected in the amount of ICG-LNP used in each patient/procedure of.
在一些实施方式中，本发明的纳米颗粒除了包括NIR荧光探针之外，还可包括至少一种可由核磁共振成像(MRI)检测的磁性探针。In some embodiments, the nanoparticles of the invention may comprise, in addition to NIR fluorescent probes, at least one magnetic probe detectable by magnetic resonance imaging (MRI).
在一些实施方式中，所述磁性探针以共价或非共价的方式结合至纳米颗粒的外表面。在其他实施方式中，磁性探针被包含在或嵌入纳米颗粒的内核中或被纳米颗粒包裹。In some embodiments, the magnetic probe is covalently or non-covalently bound to the outer surface of the nanoparticle. In other embodiments, the magnetic probes are contained within or embedded in the core of the nanoparticles or encapsulated by the nanoparticles.
磁性纳米颗粒包括永久磁性颗粒和在暴露于外部磁场之后可磁化但是当外部磁场移除时失去其磁性的那些颗粒。磁性材料或在暴露于磁场之后可磁化，当磁场移除时失去其磁性的材料是指超顺磁材料。合适的超顺磁材料的实例包括但不限于：铁、混合的铁氧化物(磁铁矿)或γ-三氧化二铁(磁赤铁矿)以及包括诸如锌之类的其他元素的取代的磁铁矿。超顺磁颗粒的尺寸可为约1nm至约20nm，例如约1nm至10nm，约5nm-20nm。Magnetic nanoparticles include permanently magnetic particles and those particles that are magnetizable after exposure to an external magnetic field but lose their magnetism when the external magnetic field is removed. Magnetic materials, or materials that can become magnetized after exposure to a magnetic field and lose their magnetism when the magnetic field is removed, are referred to as superparamagnetic materials. Examples of suitable superparamagnetic materials include, but are not limited to: iron, mixed iron oxides (magnetite) or gamma-iron seoxide (maghemite) and substituted ones including other elements such as zinc. magnetite. The size of the superparamagnetic particles may be from about 1 nm to about 20 nm, such as from about 1 nm to 10 nm, from about 5 nm to 20 nm.
超顺磁颗粒的制备以及包含这些超顺磁颗粒的纳米颗粒的制备可通过本领域已知的方法进行，例如，在De Cuyper et al.,1988,Eur Biophys J,15:311-319中所描述的方法。其他方法例如在US 7,175,912,US 7,175,909和US20050271745以及WO2014002100A1中描述，这些参考文献的全部内容通过引用并入本文。The preparation of superparamagnetic particles and the preparation of nanoparticles comprising these superparamagnetic particles can be carried out by methods known in the art, for example, in De Cuyper et al., 1988, Eur Biophys J, 15:311-319 described method. Other methods are eg described in US 7,175,912, US 7,175,909 and US20050271745 and WO2014002100A1, the entire contents of which references are incorporated herein by reference.
D.ICG-LNP的性质D. Properties of ICG-LNP
总体上，本发明提供脂质相关颗粒，例如，吲哚菁绿颗粒，其具有提高的稳定性和体内性能(参见，例如图12和图13)，用于改善淋巴管、淋巴结、淋巴异常、肿瘤和炎症的功能性高分辨率近红外荧光医学成像(参见，例如，图4-11和16-20)。In general, the present invention provides lipid-associated particles, e.g., indocyanine green particles, with improved stability and in vivo performance (see, e.g., Figures 12 and 13) for the improvement of lymphatic vessels, lymph nodes, lymphatic abnormalities, Functional high-resolution near-infrared fluorescence medical imaging of tumors and inflammation (see, eg, Figures 4-11 and 16-20).
本发明提供的颗粒所实现的荧光强度高于并超出先前含有吲哚菁绿(ICG)的脂质体或其他聚合物载体所实现的荧光强度。The particles provided by the present invention achieve fluorescence intensities that are higher than and exceed those previously achieved with indocyanine green (ICG)-containing liposomes or other polymeric carriers.
本发明提供研发用于生物相容性吲哚菁绿(ICG)纳米颗粒的独特的、简单的和可成比例扩大生产的组合物和制备方法，所述纳米颗粒使ICG强度提高4.5倍或更高，由此改善成像分辨率和灵敏度，从而使用近红外荧光(NIRF)生物成像检测被掩蔽的组织和细胞。这种4.5倍或更高的ICG荧光强度的增大是前所未有的并且是新颖的。不像先前描述的包裹有ICG的脂质体组合物和有限的脂质-ICG的偶然结合那样(所述包裹有ICG的脂质体组合物和有限的脂质-ICG的偶然结合涉及难处理的且资源密集型制备步骤)，本文所述的组合物和方法提供一种简单而又有效的三步制备程序(参见，例如，图1)，所述组合物和方法绝对稳定地将100％ICG嵌入脂质中并且在生物液体和溶液中带来潜在的最大强度。这种由简单且有效的制备程序产生的ICG荧光强度和稳定性的提高的组合(参见，例如图3，图12和图13)先前并未实现。The present invention provides the development of unique, simple and scalable compositions and preparation methods for biocompatible indocyanine green (ICG) nanoparticles that increase ICG strength by a factor of 4.5 or more High, thereby improving imaging resolution and sensitivity to detect masked tissues and cells using near-infrared fluorescence (NIRF) bioimaging. This 4.5-fold or greater increase in ICG fluorescence intensity is unprecedented and novel. Unlike previously described incidental associations of ICG-encapsulated liposome compositions and limited lipid-ICG, which involved intractable expensive and resource-intensive manufacturing steps), the compositions and methods described herein provide a simple yet efficient three-step manufacturing procedure (see, e.g., FIG. 1 ) that absolutely stably converts 100% ICG is embedded in lipids and brings potential maximum strength in biological fluids and solutions. This combination of increased ICG fluorescence intensity and stability (see, eg, Figure 3, Figure 12 and Figure 13) resulting from a simple and efficient manufacturing procedure has not been achieved previously.
在一些实施方式中，本文提供的颗粒使ICG荧光强度相对于水性ICG(13)的荧光强度提高至少2倍，3倍，4倍，4.5倍，5倍，6倍或甚至更高倍，这是任何所报道的包含脂质体的载体未实现的。In some embodiments, the particles provided herein increase the fluorescence intensity of ICG relative to the fluorescence intensity of aqueous ICG (13) by at least 2-fold, 3-fold, 4-fold, 4.5-fold, 5-fold, 6-fold or even higher, which is None of the reported liposome-containing vehicles has been achieved.
本领域技术人员已尝试将ICG包裹在蛋白质中或将ICG与蛋白质结合，但是并未实现这种荧光强度的提高以及伴随产生的本发明的稳定性(参见，例如，图11-图13)。许多科学家已尝试包裹过高浓度的ICG(10-12，14)，这实际上因荧光信号的浓度依赖性自淬灭而使ICG的荧光强度降低。Those skilled in the art have attempted to encapsulate ICG in proteins or bind ICG to proteins, but have not achieved this increase in fluorescence intensity and the concomitant stability of the invention (see, eg, Figures 11-13). Many scientists have attempted to encapsulate too high a concentration of ICG (10-12, 14), which actually reduces the fluorescence intensity of ICG due to concentration-dependent self-quenching of the fluorescent signal.
本文提供的纳米颗粒产生的ICG荧光强度的大幅度增大使光线透射穿过厚度为1cm或更厚的组织(参见，例如，图2)。这相对于游离ICG所达到的光线透射穿过的组织厚度产生了显著改善(13)，游离ICG的荧光强度使光线仅仅渗透约0.5cm的组织(参见，例如图2)。这种由荧光强度的增大而产生的提高的组织渗透深度是本发明另一非显而易见且新颖的元素。The large increase in ICG fluorescence intensity produced by the nanoparticles provided herein transmits light through tissues with a thickness of 1 cm or greater (see, eg, FIG. 2 ). This results in a significant improvement in the thickness of tissue through which light is transmitted relative to that achieved by free ICG (13), whose fluorescence intensity allows light to penetrate only about 0.5 cm of tissue (see, eg, Figure 2). This increased depth of tissue penetration resulting from an increase in fluorescence intensity is another non-obvious and novel element of the present invention.
具体而言，在美国专利公开US2014/0341813A1中提供一种使用离散剂(例如，尿素、胍、碘或其他离子)的pH-梯度方法，从而防止ICG的J-聚集并且使高浓度的单体形式的ICG(≥1mM ICG)包裹在脂质体的水性核中。然而，本领域已知，在水中，ICG在浓度为约5μM ICG的条件下开始形成聚集体(2，15-17)。虽然离散剂用于降低水分子和ICG之间的引起不稳定和聚集的相互作用，但该专利申请中所述的浓度(比导致ICG聚集的那些浓度高至少200倍)易于发生浓度依赖性淬灭并且易于损失荧光强度。实际上，虽然NIRF成像是建议的应用/领域，但是发明人注意到在第0135段中公开了“在本发明中，由于染料的累积而产生的淬灭是由抑制染料的泄露而产生的”。因此，这些颗粒中的荧光发射强度会非常有限。Specifically, a pH-gradient method using discrete agents (e.g., urea, guanidine, iodine, or other ions) is provided in U.S. Patent Publication US2014/0341813A1, thereby preventing J-aggregation of ICG and allowing high concentrations of monomer Forms of ICG (≥ 1 mM ICG) are encapsulated in the aqueous core of the liposomes. However, it is known in the art that in water, ICG begins to form aggregates at a concentration of about 5 [mu]M ICG (2, 15-17). While discrepant agents are used to reduce the destabilizing and aggregation-causing interactions between water molecules and ICG, the concentrations described in this patent application (at least 200-fold higher than those leading to ICG aggregation) are prone to concentration-dependent quenching. and are prone to loss of fluorescence intensity. Indeed, while NIRF imaging is the proposed application/area, the inventors note that in paragraph 0135 it is disclosed that "in the present invention, quenching due to dye accumulation is produced by inhibiting dye leakage" . Therefore, the intensity of fluorescence emission in these particles will be very limited.
在一些实施方式中，纳米颗粒的荧光强度是水性ICG的荧光强度的2倍至10倍。In some embodiments, the fluorescence intensity of the nanoparticles is 2 to 10 times that of aqueous ICG.
在一些实施方式中，纳米颗粒的荧光强度是水性溶液中的游离ICG的荧光强度的4倍至5倍。In some embodiments, the fluorescence intensity of the nanoparticles is 4 to 5 times that of free ICG in aqueous solution.
本文提供的纳米颗粒相对于本领域已知的颗粒具有优异的稳定性(参见，例如图11-13)。The nanoparticles provided herein have superior stability relative to particles known in the art (see, eg, Figures 11-13).
除了优化ICG-LNP制剂以实现最大荧光产量之外，相对于环境作用的稳定性也是必需的。光线使ICG产生单态氧，其通过二氧杂环丁烷反应化学分解ICG(ICG的聚甲炔链裂解成两种羰基产物，其可能具有细胞毒性)(18，19)。通常在光线充足的临床环境和手术视野中，这种光线分解是需要避免的问题。In addition to optimizing the ICG-LNP formulation for maximum fluorescence yield, stability against environmental effects is also required. Light causes ICG to generate singlet oxygen, which chemically decomposes ICG via a dioxetane reaction (the polymethine chain of ICG is cleaved into two carbonyl products, which may be cytotoxic) (18, 19). Typically in well-lit clinical environments and surgical fields, this light breakdown is a problem to be avoided.
在其目前提供的形态中，水性溶液中的游离ICG因光线而在数秒内分解。本发明公开了将ICG嵌入脂质中避免光线降解(参见，例如，图13)。ICG-LNP可在室温下暴露于光线持续若干小时并且保持几乎全部其原始荧光强度(参见，例如，图13)。In its currently offered form, free ICG in aqueous solution is decomposed within seconds by light. The present invention discloses embedding ICG in lipids to avoid photodegradation (see, eg, Figure 13). ICG-LNP can be exposed to light at room temperature for several hours and retain nearly all of its original fluorescence intensity (see, eg, Figure 13).
本文提供的纳米颗粒保持荧光强度超过其他报道(例如，室温下42天(9)以及4℃下70天(20))中的那些荧光强度(参见，例如，图12)。The nanoparticles provided herein retain fluorescence intensities that exceed those in other reports (eg, 42 days at room temperature (9) and 70 days at 4°C (20)) (see, eg, Figure 12).
在一些实施方式中，当本文提供的ICG颗粒产品在黑暗、4℃条件下储存于水性悬浮液中时，保持其荧光强度持续至少约2个月,3个月,4个月,5个月,6个月,7个月,8个月,9个月,10个月,11个月,或12个月或更长时间(参见，例如，图12)。总体而言，保持至少约50％至约60％,约50％至约65％,约50％至约70％,约50％至约75％,约50％至约80％,约50％至约85％,约50％至约90％,约50％至约95％,或约50％至约100％的荧光。In some embodiments, the ICG particle product provided herein retains its fluorescence intensity for at least about 2 months, 3 months, 4 months, 5 months when stored in an aqueous suspension in the dark at 4°C , 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months or more (see, eg, Figure 12). Generally, maintaining at least about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to About 85%, about 50% to about 90%, about 50% to about 95%, or about 50% to about 100% fluorescent.
在一些实施方式中，本文提供的ICG-LNP在血清中稳定，并且在25℃下在热灭活的大鼠血清中6小时后保持其初始荧光的约50％至约60％,约50％至约65％,约50％至约70％,约50％至约75％,约50％至约80％,约50％至约85％,约50％至约90％,约50％至约95％,或约50％至约100％。在一些实施方式中，本文提供的ICG-LNP在血清中稳定，并且在25℃下，在热灭活的大鼠血清中6小时后保持其初始荧光的94±0.6％。In some embodiments, the ICG-LNP provided herein is stable in serum and retains about 50% to about 60%, about 50%, of its initial fluorescence after 6 hours at 25°C in heat-inactivated rat serum to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, or about 50% to about 100%. In some embodiments, the ICG-LNP provided herein is stable in serum and retains 94±0.6% of its initial fluorescence after 6 hours in heat-inactivated rat serum at 25°C.
在一些实施方式中，本文提供的ICG-LNP在血清中稳定，并且在热灭活的大鼠血清中持续一段时间段保持其初始荧光的至少约90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,或100％，所述时间段例如约1hr,2hr,3hr,4hr,5hr,6hr,8hr,10hr,12hr,14hr,16hr,18hr,20hr,24hr或更长时间。In some embodiments, the ICG-LNP provided herein is stable in serum and retains at least about 90%, 91%, 92%, 93% of its initial fluorescence for a period of time in heat-inactivated rat serum, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for example about 1hr, 2hr, 3hr, 4hr, 5hr, 6hr, 8hr, 10hr, 12hr, 14hr, 16hr, 18hr, 20hr, 24hr or more.
在一些实施方式中，在黑暗中4℃下储存超过300天之后，纳米颗粒的荧光强度是其初始荧光强度的50％至100％。In some embodiments, the fluorescence intensity of the nanoparticles is 50% to 100% of their initial fluorescence intensity after storage at 4°C in the dark for more than 300 days.
在一些实施方式中，纳米颗粒的平均半衰期为0.5hr至1hr，0.5hr至2hr,0.5hr至3hr,0.5hr至4hr,0.5hr至5hr,0.5hr至6hr,0.5hr至7hr,0.5hr至8hr,0.5hr至9hr,或0.5hr至10hr或者更长时间。In some embodiments, the nanoparticles have an average half-life of 0.5 hr to 1 hr, 0.5 hr to 2 hr, 0.5 hr to 3 hr, 0.5 hr to 4 hr, 0.5 hr to 5 hr, 0.5 hr to 6 hr, 0.5 hr to 7 hr, 0.5 hr to 8hr, 0.5hr to 9hr, or 0.5hr to 10hr or longer.
本文提供的ICG颗粒产品的进一步稳定通过用诸如海藻糖或蔗糖之类的冷冻保护剂的冷冻干燥法实现，所述冷冻保护剂在ICG颗粒产品与水水合之后维持颗粒特性。Further stabilization of the ICG particulate products provided herein is achieved by freeze-drying with cryoprotectants such as trehalose or sucrose that maintain particle properties after hydration of the ICG particulate products with water.
综上所述，所观察到的光线、储存和血清稳定性表明ICG-LNP具有很强的临床使用稳定性，尤其是用于淋巴系统的成像，所述淋巴系统中，淋巴包含的血清蛋白浓度比血液中的血清蛋白浓度低大约两倍。Taken together, the observed light, storage, and serum stability suggest that ICG-LNP has strong stability for clinical use, especially for imaging the lymphatic system, where the lymph contains serum protein concentrations About two times lower than the serum protein concentration in the blood.
本文提供的LNP由于其较高的荧光强度而比本领域已知的其他ICG试剂更加有效。本文中的“有效”是指在施用于组织或器官之后能够通过相同或更少的ICG分子发射更高的荧光，从而产生图像用于期望的目的。The LNPs provided herein are more potent than other ICG reagents known in the art due to their higher fluorescence intensity. "Effective" herein means capable of emitting higher fluorescence by the same or fewer ICG molecules after application to a tissue or organ, thereby generating an image for the desired purpose.
在一些实施方式中，组合物按单位体重给药剂量在小于约0.5mg/kg,0.4mg/kg,0.3mg/kg,0.2mg/kg,0.1mg/kg,0.09mg/kg,,0.08mg/kg,0.07mg/kg,0.06mg/kg,0.05mg/kg,0.04mg/kg,0.03mg/kg,0.02mg/kg,或0.01mg/kgICG剂量的条件下有效。In some embodiments, the composition is administered at a dose per body weight of less than about 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, 0.1 mg/kg, 0.09 mg/kg, 0.08 mg /kg, 0.07mg/kg, 0.06mg/kg, 0.05mg/kg, 0.04mg/kg, 0.03mg/kg, 0.02mg/kg, or 0.01mg/kg ICG doses are effective.
在一些实施方式中，组合物按单位体重给药剂量在约0.01mg/kg至约0.5mg/kg,约0.01mg/kg至约0.4mg/kg,约0.01mg/kg至约0.3mg/kg,约0.01mg/kg至约0.2mg/kg,约0.01mg/kg至约0.1mg/kg,约0.01mg/kg至约0.09mg/kg,约0.01mg/kg至约0.08mg/kg,约0.01mg/kg至约0.07mg/kg,约0.01mg/kg至约0.06mg/kg,约0.01mg/kg至约0.05mg/kg,约0.01mg/kg至约0.04mg/kg,约0.01mg/kg至约0.03mg/kg,或约0.01mg/kg至约0.02mg/kg ICG剂量(包括端点值)的条件下有效。In some embodiments, the composition is administered at a dose per unit body weight of about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 0.4 mg/kg, about 0.01 mg/kg to about 0.3 mg/kg , about 0.01 mg/kg to about 0.2 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about 0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 0.04 mg/kg, about 0.01 mg /kg to about 0.03mg/kg, or about 0.01mg/kg to about 0.02mg/kg ICG doses (endpoints included).
在一些实施方式中，组合物在约0.05mg/kg至0.5mg/kg ICG剂量(包括端点值)的条件下有效。In some embodiments, the compositions are effective at an ICG dose of about 0.05 mg/kg to 0.5 mg/kg, inclusive.
本文提供的组合物或ICG-LNP的有效性通过使用本领域已知的方法测量。例如，在公开的文献值中，比较纳米颗粒相对于水性ICG的荧光增强倍数。The effectiveness of the compositions provided herein or ICG-LNP is measured using methods known in the art. For example, in published literature values, compare the fluorescence enhancement factor of nanoparticles relative to aqueous ICG.
脂质体的一种熟知特性是它们在肝脏组织中积累(7)。然而，本发明的ICG-LNP不在肝脏中积累，但是其通过肝胆管几乎被完全清除而进入肠道，这是这些ICG颗粒的不同之处和新颖之处(参见，例如，图19)。A well-known property of liposomes is their accumulation in liver tissue (7). However, the ICG-LNP of the present invention does not accumulate in the liver, but is almost completely cleared into the gut via the hepatic bile ducts, which is different and novel for these ICG particles (see, eg, Figure 19).
本文提供的ICG颗粒比任何报道过的组合物和方法都更加稳定。而且，这些颗粒可用于识别肿瘤(参见，例如，图4)和带有淋巴血管形成的炎症位点(参见，例如，图5-8和图20)。本发明研发的颗粒稳定且特异性地将NIRF分子传送至淋巴系统以进行NIRF成像。The ICG particles provided herein are more stable than any reported compositions and methods. Furthermore, these particles can be used to identify tumors (see, eg, Figure 4) and sites of inflammation with lymphovascularization (see, eg, Figures 5-8 and Figure 20). The particles developed in the present invention stably and specifically deliver NIRF molecules to the lymphatic system for NIRF imaging.
II.制备ICG-LNP的方法II. Method for preparing ICG-LNP
另一方面，本发明提供一种解决ICG的水不稳定性、聚集和降解的限制的简单方案并且使更深层的组织水平以比单独使用ICG更好的分辨率成像。在本发明提供的方法中，由于ICG100％(或大约100％)嵌入脂质中，因此不需要分离步骤，强ICG-脂质结合相互作用通过简单的三步制备程序产生(参见，例如，图1)，并且ICG自淬灭被降低以最大程度增大ICG荧光强度产量(参见，例如，图3)。On the other hand, the present invention provides a simple solution to address the limitations of ICG's water instability, aggregation and degradation and enables imaging of deeper tissue levels with better resolution than ICG alone. In the methods provided by the present invention, since ICG is 100% (or approximately 100%) embedded in lipids, no isolation step is required, and strong ICG-lipid binding interactions are produced by a simple three-step preparation procedure (see, e.g., Fig. 1), and ICG self-quenching was reduced to maximize ICG fluorescence intensity yield (see, eg, Figure 3).
本发明提供一种仅涉及三个步骤的简单/流畅的制备方法：(1)在有机溶剂中混合吲哚菁绿(ICG)与脂质分子，(2)蒸发含有完全混合的脂质和ICG的有机溶剂，以及(3)降低水合的ICG-脂质混合物的粒度，并且由于ICG完全嵌入ICG-脂质纳米颗粒而不需要过滤/纯化/分离步骤除去游离的未合并的ICG(参见，例如，图1)。The present invention provides a simple/fluid preparation method involving only three steps: (1) mixing indocyanine green (ICG) with lipid molecules in an organic solvent, (2) evaporating organic solvent, and (3) reduce the particle size of the hydrated ICG-lipid mixture and remove free unincorporated ICG without the need for filtration/purification/isolation steps since ICG is fully embedded in ICG-lipid nanoparticles (see, e.g. ,figure 1).
在一些实施方式中，制备纳米颗粒的方法包括：(a)在有机溶剂中混合吲哚菁绿(ICG)与脂质分子；(b)蒸发有机溶剂以产生含有所述脂质分子和所述ICG的薄膜；(c)使用缓冲盐水水合所述薄膜；(d)使用超声能量或均质步骤减小粒度，从而减小用所述缓冲盐水水合的薄膜的粒度。In some embodiments, the method of preparing nanoparticles comprises: (a) mixing indocyanine green (ICG) and lipid molecules in an organic solvent; (b) evaporating the organic solvent to produce a compound containing the lipid molecules and the Films of ICG; (c) hydrating the film using buffered saline; (d) reducing the particle size using ultrasonic energy or a homogenization step, thereby reducing the particle size of the film hydrated with the buffered saline.
本文提供的方法与本领域已知的方法不同，本领域已知的方法忽略了在形成脂质薄膜之前的作为初始制备步骤的在有机溶剂中混合ICG和脂质，该步骤是将ICG稳定地嵌入脂质所必须的。相反，本领域已知的方法将ICG的水性溶液加至已形成的脂质薄膜中，这导致ICG无法有效包裹至脂质体的水性核中。The method provided herein differs from methods known in the art which omit the mixing of ICG and lipids in an organic solvent prior to the formation of lipid films as an initial preparation step that stabilizes ICG Necessary for embedding lipids. In contrast, the methods known in the art add an aqueous solution of ICG to the formed lipid film, which results in the ineffective encapsulation of ICG into the aqueous core of liposomes.
在一些实施方式中，脂质分子包括1,2-二硬脂酰基-sn-丙三基-3-磷酸胆碱(DSPC)和1,2-二硬脂酰基-sn-丙三基-3-磷酸乙醇胺-N-甲氧基-聚乙二醇-2000(DSPEmPEG2000)。In some embodiments, the lipid molecule comprises 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glyceryl-3 - Phosphoethanolamine-N-methoxy-polyethylene glycol-2000 (DSPEmPEG2000).
在一些实施方式中，有机溶剂包括乙醇、甲醇、氯仿、乙酸乙酯或DMSO。In some embodiments, the organic solvent includes ethanol, methanol, chloroform, ethyl acetate, or DMSO.
在一些实施方式中，脂质纳米颗粒悬浮于包含生物相容性缓冲液的盐组合物中，所述生物相容性缓冲液例如pH为5-8且Osm为约303mOSM的缓冲液。这些缓冲液的实例包括生理盐水、林格氏溶液、5％葡萄糖水溶液或0.9％NaCl和约20mM NaHCO3的缓冲液。In some embodiments, the lipid nanoparticles are suspended in a salt composition comprising a biocompatible buffer, eg, a buffer with a pH of 5-8 and an Osm of about 303 mOSM. Examples of these buffers include physiological saline, Ringer's solution, 5% dextrose in water, or a buffer of 0.9% NaCl and about 20 mMNaHCO3 .
在一些实施方式中，缓冲盐水包括约0.9％NaCl和约20mM NaHCO3。In some embodiments, buffered saline includes about 0.9% NaCl and about 20 mM NaHCO3 .
在一些实施方式中，所述方法不包括过滤步骤，纯化步骤或分离步骤，或者它们的组合。In some embodiments, the method does not include a filtration step, purification step, or isolation step, or a combination thereof.
在一些实施方式中，所述方法不包括任何过滤步骤、纯化步骤或分离步骤。In some embodiments, the method does not include any filtration, purification or isolation steps.
美国专利公开US2014/0341813A1公开了一种使用离散剂(例如，尿素、胍、碘或其他离子)的pH梯度方法以防止ICG的J-聚集并使高浓度的单体形式的ICG(≥1mM ICG)包裹在脂质体的水性核中。然而，该引用的专利申请中所描述的制备步骤鉴于下述多种其他原因而非常繁琐(≥5个步骤)，所述原因包括需要使用pH-梯度，ICG在水性溶液中不稳定(虽然存在离散剂)以及由于所有游离ICG的不完全包裹而需要进行过滤和纯化(该美国专利公开中报道了仅27.4％的包裹率)。U.S. Patent Publication US2014/0341813A1 discloses a pH gradient method using discrete agents (e.g., urea, guanidine, iodine, or other ions) to prevent J-aggregation of ICG and allow high concentrations of ICG in monomeric form (≥1 mM ICG ) encapsulated in the aqueous core of liposomes. However, the preparation steps described in this cited patent application are very tedious (≥ 5 steps) for a number of other reasons including the need to use a pH-gradient, the instability of ICG in aqueous solutions (although there are discrete agent) and the need for filtration and purification due to incomplete entrapment of all free ICG (only 27.4% entrapment reported in this US patent publication).
本文公开的制备方法简单得多，因为其不涉及pH-梯度或纯化步骤，其仅仅涉及三个简单步骤：混合、干燥和水合/尺寸减小。The preparation method disclosed here is much simpler as it does not involve pH-gradients or purification steps, it only involves three simple steps: mixing, drying and hydration/size reduction.
在一些实施方式中，脂质纳米颗粒悬浮于包含约0.9％NaCl和约5-50mM(例如，5,10,15,20,25,30,35,40,45,或50mM)的NaHCO3的盐组合物中。In some embodiments, the lipid nanoparticles are suspended in a salt comprising about 0.9% NaCl and about 5-50 mM (e.g., 5, 10, 15, 20, 25,30 , 35, 40, 45, or 50 mM) NaHCO composition.
本文提供的方法和组合物基于意想不到地发现了简单且可成比例扩大生产的3步制备方法和组合物，其能够使吲哚菁绿(ICG)几乎100％地嵌入脂质以形成稳定的脂质纳米颗粒(LNP)。The methods and compositions provided herein are based on the unexpected discovery of a simple and scalable 3-step preparation and composition that enables nearly 100% lipid intercalation of indocyanine green (ICG) to form stable Lipid Nanoparticles (LNPs).
这种使ICG与脂质结合的独一无二的方法极大地提高了ICG的稳定性和近红外荧光(NIRF)强度，超过了游离ICG所能达到的稳定性和近红外荧光强度，这使更深层的组织能够成像。虽然本发明的LNP制剂类似于脂质体制剂，但是本发明公开了新的特性，这在早期的脂质体生理化学研究和体内研究中是非显而易见的，并且这些ICG-LNP表现出比其他已报道的ICG脂质体更加优异的特性，即，(1)100％嵌入效率，这排除了任何纯化步骤，(2)产生高稳定性颗粒的简单的3步制备步骤，以及(3)优化ICG光谱学性质从而避免ICG荧光发射的自淬灭并极大地提高ICG的荧光强度产量。将这些要素综合在一个具有高度特异性组成的系统中是非显而易见的并且提供了前所未有的增加体内NIR成像时间和分辨行为的协同作用。具体而言，本发明的组合物特别适于用于淋巴结构和淋巴网络绘图，识别淋巴病理学和淋巴异常，识别带有淋巴血管形成的肿瘤，识别带有三级淋巴器官的炎症位点，以及将药物递送至淋巴系统以及与淋巴相关的病理学的淋巴管和淋巴结中的摄取、留滞和广泛分布。This unique method of binding ICG to lipids dramatically increases ICG stability and near-infrared fluorescence (NIRF) intensity beyond that achieved with free ICG, which enables deeper Tissues can be imaged. Although the LNP formulations of the present invention are similar to liposomal formulations, the present invention discloses novel properties that were not apparent from earlier liposomal physiochemical studies and in vivo studies, and these ICG-LNPs exhibited greater Further superior properties of the reported ICG liposomes, namely, (1) 100% intercalation efficiency, which eliminates any purification steps, (2) a simple 3-step preparation procedure yielding highly stable particles, and (3) optimized ICG The spectroscopic properties thus avoid self-quenching of ICG fluorescence emission and greatly increase the fluorescence intensity yield of ICG. Integrating these elements in a system of highly specific composition is non-obvious and offers an unprecedented synergy of increasing in vivo NIR imaging time and resolving behavior. In particular, the composition of the invention is particularly suitable for mapping lymphatic structures and lymphatic networks, identifying lymphatic pathology and lymphatic abnormalities, identifying tumors with lymphatic angiogenesis, identifying sites of inflammation with tertiary lymphoid organs, As well as uptake, retention and widespread distribution in lymphatic vessels and lymph nodes for drug delivery to the lymphatic system and lymphatic-related pathologies.
本发明提供的纳米颗粒的制备方法提供了相对于本领域已知方法的各种新颖的方面和优势，例如：(1)综合组合物和三步制备方法生成物理上稳定的带有独特性质的产品(稳定且高ICG荧光产量)，(2)综合ICG光谱学性质提高效力，(3)避免游离ICG的除去的简单三步过程，这降低了总体成本并且控制由于复杂的多步过程而产生的潜在污染，(4)荧光分子(吲哚菁绿(ICG))嵌入ICG-脂质纳米颗粒(ICG-LNP)中，这提高了荧光强度，使其大大超过包裹在脂质体或脂质囊泡中的水性ICG所能达到的荧光强度，(5)在嵌入脂质的组合物中使用的ICG剂量相对于用于体内近红外(NIR)成像的水性ICG(临床使用)降低了约10倍，这产生了更高的分辨率和荧光强度，以及(6)使ICG几乎100％嵌入脂质膜中的独特的组成、尺寸和表面性质，提高的稳定性(储存和体内这两方面)，以及对于淋巴系统的体内功能性医学成像而言非常重要的性能。The preparation method of nanoparticles provided by the present invention provides various novel aspects and advantages over methods known in the art, such as: (1) comprehensive composition and three-step preparation method to generate physically stable nanoparticle with unique properties product (stable and high ICG fluorescence yield), (2) integrated ICG spectroscopic properties enhance potency, (3) simple three-step process avoiding removal of free ICG, which reduces overall cost and controls generation due to complex multi-step process (4) Fluorescent molecules (indocyanine green (ICG)) embedded in ICG-lipid nanoparticles (ICG-LNP), which enhanced the fluorescence intensity, making it much higher than those encapsulated in liposomes or lipids Fluorescence intensities achievable with aqueous ICG in vesicles, (5) ICG doses used in lipid-embedded compositions were reduced by about 10% relative to aqueous ICG for in vivo near-infrared (NIR) imaging (clinical use) times, which yielded higher resolution and fluorescence intensity, and (6) unique composition, size, and surface properties that enable ICG to be almost 100% embedded in lipid membranes, improving stability (both in storage and in vivo) , and a very important property for in vivo functional medical imaging of the lymphatic system.
III.药物制剂和给药方式III. Drug Formulation and Administration
本发明还涉及包含本发明的化合物或其药学上可接受的盐以及一种或多种药学上可接受的载体的药物制剂。The present invention also relates to pharmaceutical formulations comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
A.对比剂A. Contrast agent
本文提供的纳米颗粒可用作荧光成像或光声成像的对比剂，因为该颗粒包含ICG并且可吸收近红外光以发射荧光或声波。此外，该颗粒可用作视觉检查的对比剂，因为ICG具有绿色。The nanoparticles provided herein can be used as contrast agents for fluorescence imaging or photoacoustic imaging because the particles contain ICG and can absorb near-infrared light to emit fluorescence or acoustic waves. In addition, the particles can be used as a contrast agent for visual inspection, since ICG has a green color.
本文中的“对比剂”是指能够在想要观察的组织或分子，存在于标本中的组织或分子和所述组织或分子周围的组织或分子之间进行比较产生不同以改善想要观察的组织或分子的形态信息或位置信息的检测灵敏度的物质。"Contrast agent" herein refers to a tissue or molecule that can be compared between the tissue or molecule to be observed, the tissue or molecule present in the specimen, and the tissue or molecule around the tissue or molecule to produce a difference to improve the tissue or molecule to be observed. Substances that are sensitive to detection of morphological information or positional information of tissues or molecules.
本文中的“荧光成像”或“光声成像”是指通过例如荧光检测设备或光声信号检测设备成像的组织或分子。"Fluorescence imaging" or "photoacoustic imaging" herein refers to tissue or molecules imaged by, for example, a fluorescence detection device or a photoacoustic signal detection device.
根据本实施方式的对比剂包括根据本文提供的颗粒和所述颗粒分散于其中的分散介质。所述分散介质是用于分散颗粒的液体物质并且其实例包括生理盐水和注射用蒸馏水。此外，对比剂可具有药理学上可接受的诸如食盐或葡萄糖之类的添加剂。所述对比剂可以是如下对比剂：根据本实施方式的颗粒预先分散于分散介质，或者可如下描述的那样使用。根据本实施方式的颗粒和分散介质包装在试剂盒中，并且所述颗粒在给药于活体(例如，人体)之前分散于分散介质。The contrast agent according to the present embodiment comprises the particles according to provided herein and a dispersion medium in which the particles are dispersed. The dispersion medium is a liquid substance for dispersing particles and examples thereof include physiological saline and distilled water for injection. In addition, contrast media may have pharmacologically acceptable additives such as common salt or glucose. The contrast agent may be a contrast agent in which particles according to the present embodiment are previously dispersed in a dispersion medium, or may be used as described below. The particles and dispersion medium according to the present embodiment are packaged in a kit, and the particles are dispersed in the dispersion medium before administration to a living body (for example, a human body).
IV.本发明的试剂盒IV. The kit of the present invention
关于组合物的使用的说明总体上包括用于目标治疗的剂量、给药方案和给药途径的信息。组合物的容器可以是单位剂量，批量包装(例如，多剂量包装)或子单位剂量。在本发明的试剂盒中提供的说明书通常是在标签上或包装插入物上(例如，包括在试剂盒中的纸页)的书面说明，但是机器可读的说明书(例如，磁盘或光盘中载有的说明)也是可接受的。Instructions for the use of the compositions generally include information on dosages, dosing regimens and routes of administration for the intended treatment. The containers of the compositions can be unit doses, bulk packs (eg, multi-dose packs) or sub-unit doses. The instructions provided in the kits of the invention are typically written instructions on a label or on a package insert (e.g., a paper sheet included with the kit), but machine-readable instructions (e.g., Some instructions) are also acceptable.
V.医药用途V. Medical use
大多数荧光医学成像(小于600nm-700nm)受到体内血液、组织和脂肪的光吸收和光散射的限制。因为吲哚菁绿(ICG)在820nm发射荧光，其在血液、水、组织和脂质的光吸收系数最低的700nm-900nm的范围的中间，因此该近红外(NIR)化合物克服了生物组织的干扰。Most fluorescent medical imaging (less than 600nm-700nm) is limited by light absorption and light scattering by blood, tissue and fat in vivo. Because indocyanine green (ICG) emits fluorescence at 820nm, which is in the middle of the range of 700nm-900nm where the light absorption coefficients of blood, water, tissue and lipids are lowest, this near-infrared (NIR) compound overcomes the interference.
皮下注射之后，稳定地嵌入ICG-LNP中的ICG被优化为保留在淋巴系统中，而不会如游离ICG那样从淋巴管中渗出进入周围组织(参见，例如，图8和图9)。Following subcutaneous injection, ICG stably embedded in ICG-LNP is optimized to remain in the lymphatic system without leaking out of the lymphatic vessels into surrounding tissues like free ICG does (see, eg, Figures 8 and 9).
ICG-LNP可由临床医师使用，用于从手术至诊断和药物递送的一系列临床应用中。具体而言，ICG-LNP可用于在手术中识别淋巴管和淋巴结(LN)(参见，例如，图9和图10)，用于识别淋巴异常(参见，例如，图5-8)，使实体肿瘤中的前哨淋巴结显现，使带有淋巴血管形成的肿瘤显现(参见，例如图4)，使带有三级淋巴器官的炎症位点显现(参见，例如，图5-8)，将药物递送至淋巴管、淋巴结和病变位置。ICG-LNP can be used by clinicians in a range of clinical applications from surgery to diagnosis and drug delivery. Specifically, ICG-LNP can be used to identify lymphatic vessels and lymph nodes (LNs) intraoperatively (see, eg, Figures 9 and 10), to identify lymphatic abnormalities (see, eg, Figures 5-8), to enable solid Visualization of sentinel lymph nodes in tumors, visualization of tumors with lymphatic vascularization (see, eg, Figure 4), visualization of sites of inflammation with tertiary lymphoid organs (see, eg, Figures 5-8), drug delivery to lymphatic vessels, lymph nodes and lesion sites.
更加具体而言，ICG是唯一由美国食品和药品管理局(FDA)和欧洲药物管理局(EMA)批准的用于人体的NIR荧光团。为了使淋巴脉管系统成像，目前临床上通过将纯ICG溶质溶解于水溶液并进行皮下注射来使用ICG。ICG与白蛋白的高结合亲和力能够使其被摄取至淋巴管中，然而，这种白蛋白结合不稳定并且是可逆的，ICG从白蛋白中解离并由于浓度梯度驱动力从淋巴毛细管中渗漏出来，而暴露于使ICG降解的水性环境。通过将ICG稳定地嵌入LNP的脂质中，这(1)通过不可逆的脂质结合稳定ICG，(2)提高其荧光产量和高荧光强度的持续期，以及(3)避免ICG从颗粒中解离和由于脂质纳米颗粒的尺寸而从淋巴脉管系统中漏出。More specifically, ICG is the only NIR fluorophore approved for use in humans by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). To image lymphatic vasculature, ICG is currently used clinically by dissolving pure ICG solutes in aqueous solution and injecting them subcutaneously. The high binding affinity of ICG to albumin enables its uptake into lymphatic vessels; however, this albumin binding is unstable and reversible, ICG dissociates from albumin and permeates from lymphatic capillaries due to a concentration gradient-driven force. Leakage, and exposure to an aqueous environment that degrades ICG. By stably embedding ICG into the lipids of LNPs, this (1) stabilizes ICG through irreversible lipid binding, (2) increases its fluorescence yield and duration of high fluorescence intensity, and (3) avoids the dissolution of ICG from particles. Dissociation and leakage from lymphatic vasculature due to the size of lipid nanoparticles.
ICG-LNP保留在远离注射位点的下游的淋巴脉管系统中并且在淋巴结(LN)中累积，这产生对淋巴系统具有特异性的且比临床使用的游离ICG组合物和其他ICG纳米颗粒制剂(例如包裹在脂质体的水性核中的ICG(8-12，21)(US专利公开US2014/0341813A1))显著更强和更稳定的广泛的高分辨率NIR荧光成像。ICG-LNP is retained in the lymphatic vasculature downstream from the injection site and accumulates in the lymph node (LN), which results in a free ICG composition and other ICG nanoparticle formulations that are specific to the lymphatic system and more clinically used (eg ICG(8-12,21) encapsulated in the aqueous core of liposomes (US Patent Publication US2014/0341813A1 )) Significantly stronger and more stable broad-based high-resolution NIR fluorescence imaging.
而且，本发明公开的简单且有效的制备步骤不涉及任何高成本和耗时的分离或纯化步骤，并且使几乎100％的ICG嵌入脂质，其对于大规模生产是有用的。Moreover, the simple and efficient preparation steps disclosed in the present invention do not involve any costly and time-consuming isolation or purification steps and enable almost 100% ICG intercalation into lipids, which is useful for large-scale production.
因为由本发明的独特的且简单的3-步制备方法和组合物而产生颗粒稳定性，因此，产生了淋巴结构和血管结构的一致的且广泛的高分辨率成像(参见，例如图5-10，16，19和20)，所述成像可用于淋巴系统的疾病的诊断、评估和监控以及区分正常与异常淋巴结构和功能。全身血液分布可使用这些颗粒维持，从而检测高度灌注的组织和器官(例如，肝脏)中的组织异常(参见，例如图18)。Because of the particle stability resulting from the unique and simple 3-step preparation method and composition of the present invention, consistent and extensive high-resolution imaging of lymphoid and vascular structures is produced (see, e.g., FIGS. 5-10 , 16, 19 and 20), the imaging can be used in the diagnosis, assessment and monitoring of diseases of the lymphatic system and in distinguishing normal from abnormal lymphoid structure and function. Systemic blood distribution can be maintained using these particles, thereby detecting tissue abnormalities in highly perfused tissues and organs (eg, the liver) (see, eg, FIG. 18 ).
一方面，本发明提供具有临床用于人体NIRF成像的产品(ICG)的促进剂/稳定剂的纳米颗粒，这样，本发明的技术可用于提高ICG的稳定性和荧光强度，从而改善已有NIR成像仪器的NIRF成像。In one aspect, the present invention provides nanoparticles with accelerators/stabilizers for products (ICG) clinically used in human NIRF imaging, so that the technology of the present invention can be used to improve the stability and fluorescence intensity of ICG, thereby improving existing NIR NIRF imaging of imaging instruments.
ICG-LNP用作改善NIRF成像能力的新型诊断成像工具以在受治者体内(例如人类)识别/显现(1)手术过程中的淋巴管和血管以及结节，(2)淋巴和血管异常，(3)实体肿瘤的前哨淋巴结，(4)带有淋巴血管形成或总血管形成的肿瘤，以及(5)带有三级淋巴器官的炎症位点或组织中的炎症位点。ICG-LNP is used as a novel diagnostic imaging tool to improve the ability of NIRF imaging to identify/visualize (1) lymphatic and blood vessels and nodules during surgery, (2) lymphatic and vascular abnormalities, (3) sentinel lymph nodes of solid tumors, (4) tumors with lymphangiogenesis or gross vasculature, and (5) sites of inflammation with tertiary lymphoid organs or in tissues.
本文中的“受治者”是指人类或非人类动物，包括但不限于：诸如狗、猫、马、牛、猪、兔、豚鼠、绵羊、山羊、灵长类动物、大鼠和小鼠之类的哺乳动物。"Subject" herein refers to human or non-human animals, including but not limited to: such as dogs, cats, horses, cows, pigs, rabbits, guinea pigs, sheep, goats, primates, rats and mice such mammals.
此外，ICG-LNP与其他诸如表6所列的分子之类的疏水分子的结合可用于提高它们的稳定性和用于递送药物至淋巴管、淋巴结和病变位置。In addition, conjugation of ICG-LNP with other hydrophobic molecules such as those listed in Table 6 can be used to increase their stability and for drug delivery to lymphatic vessels, lymph nodes and lesion sites.
在治疗淋巴疾病中，NIRF成像是广受欢迎的指导诊断和治疗的工具。本发明公开的ICG-LNP的使用通过在临床环境中使NIRF成像的强度和实用性多元化和得到改进而改善了NIRF成像能力。In the treatment of lymphoid diseases, NIRF imaging is a popular tool to guide diagnosis and treatment. The use of ICG-LNP disclosed herein improves NIRF imaging capabilities by diversifying and improving the strength and utility of NIRF imaging in clinical settings.
本文提供的ICG-LNP具有各种各样的比现有技术中已知的ICG制剂更加优异的有利性质，例如(1)在皮下注射之后被快速摄取至淋巴中(参见，例如表5，图17)，(2)在整个淋巴系统中快速流动和广泛分布(参见，例如图9和图10)，(3)在第一次流过淋巴系统之后滞留于淋巴管和淋巴结中(参见，例如，图11)，(4)在淋巴系统中具有非常高的体内稳定性(参见，例如，图9-11)，(5)在淋巴结组织中稳定，如在淋巴结组织切片的近红外显微成像下看到的通过间断荧光所显示的(参见，例如，图11)，(6)分布于淋巴结组织中，如淋巴结组织切片的近红外显微成像所显示的(参见，例如，图11)，(7)相对于游离ICG，使用脂质-结合ICG的不同的/提高的淋巴管和淋巴结的高分辨率成像(参见，例如，表2和图10)，(8)检测注射位点的下游淋巴结，这是游离ICG无法实现的(参见，例如，表2，表5，图10，17，19)，(9)相对于游离ICG，使用脂质-结合的ICG使淋巴管和淋巴结成像的持续期延长(图10、19)，(10)在淋巴结中累积和截留，尤其是在病变的淋巴结中累积和截留(图6，8，11，19，20)，(11)检测带有淋巴血管形成的肿瘤(图4)，(12)检测由于淋巴管新生和三级淋巴器官而产生的炎症位点(图5-8)，和(13)主要通过胆管从肝脏清除进入肠道(图18)。The ICG-LNP provided herein has a variety of advantageous properties that are superior to ICG formulations known in the prior art, such as (1) rapid uptake into the lymph after subcutaneous injection (see, e.g., Table 5, Fig. 17), (2) rapid flow and widespread distribution throughout the lymphatic system (see, e.g., Figures 9 and 10), (3) retention in lymphatic vessels and lymph nodes after first flow through the lymphatic system (see, e.g. , Figure 11), (4) have very high in vivo stability in the lymphatic system (see, for example, Figures 9-11), (5) are stable in lymph node tissue, as shown in near-infrared microscopy imaging of lymph node tissue sections Seen below as shown by intermittent fluorescence (see, e.g., FIG. 11 ), (6) distributed in lymph node tissue, as shown by near-infrared microscopic imaging of lymph node tissue sections (see, e.g., FIG. 11 ), (7) High resolution imaging of different/enhanced lymphatic vessels and lymph nodes using lipid-conjugated ICG relative to free ICG (see, e.g., Table 2 and Figure 10), (8) detection downstream of the injection site Lymph nodes, which is not possible with free ICG (see, e.g., Table 2, Table 5, Figures 10, 17, 19), (9) Imaging of lymphatic vessels and lymph nodes using lipid-bound ICG versus free ICG Prolonged persistence (Fig. 10, 19), (10) accumulation and entrapment in lymph nodes, especially in diseased lymph nodes (Fig. 6, 8, 11, 19, 20), (11) detection of Tumors that formed blood vessels (Fig. 4), (12) detected sites of inflammation due to lymphangiogenesis and tertiary lymphoid organs (Fig. 18).
A.荧光成像方法A. Fluorescent Imaging Methods
本文提供的对比剂还可用于荧光成像方法。使用根据本实施方式的对比剂的荧光成像方法包括至少下列步骤：将对比剂给药于受治者或获自受治者的样本，用光辐照受治者或获自受治者的样本，以及测量来自于从受治者或获自受治者的样本中存在的颗粒中衍生的物质的荧光。The contrast agents provided herein can also be used in fluorescence imaging methods. The fluorescence imaging method using the contrast agent according to the present embodiment includes at least the following steps: administering a contrast agent to a subject or a sample obtained from the subject, irradiating the subject or the sample obtained from the subject with light , and measuring fluorescence from substances derived from particles present in the subject or a sample obtained from the subject.
使用根据本实施方式的对比剂的荧光成像方法的实例在下文中描述。即，将根据本实施方式的对比剂施加于标本，或加至诸如获自标本的器官之类的样本。应当注意的是，所述标本是指所有活体，例如，实验动物和宠物，没有任何特定限制。标本或获自标本的样本的实例可包括器官、组织、组织切片，细胞和细胞裂解物。在施加颗粒或添加颗粒之后，用近红外波长范围内的光辐照所述标本或类似物。An example of a fluorescence imaging method using a contrast agent according to the present embodiment is described below. That is, the contrast agent according to the present embodiment is applied to the specimen, or to a sample such as an organ obtained from the specimen. It should be noted that the specimens refer to all living bodies, for example, experimental animals and pets, without any specific limitation. Examples of specimens or samples obtained from specimens may include organs, tissues, tissue sections, cells and cell lysates. After applying the particles or adding the particles, the specimen or the like is irradiated with light in the near-infrared wavelength range.
成像可通过商售荧光IVIS成像设备(例如，IVIS Lumina成像系统)和ICG滤波器进行。Imaging can be performed with commercially available fluorescent IVIS imaging equipment (eg, IVIS Lumina Imaging System) and ICG filters.
如上所述，许多光成像设备被设计为使用波长范围并且IVIS也对应于ICG单体的吸收波长，即，780nm(滤波器的激发通带设定为4:705至780nm)。As mentioned above, many optical imaging devices are designed to use the wavelength range and the IVIS also corresponds to the absorption wavelength of the ICG monomer, ie, 780 nm (the excitation passband of the filter is set at 4: 705 to 780 nm).
一方面，本发明提供一种用于使受治者体内的组织或器官成像的方法，包括：将适量的ICG-LNP给药于需要成像的受治者，用合适频率的光辐照所述受治者，以及通过检测ICG发射的光获取所述受治者的组织或器官的图像。Nguyen,Q.T.,and Tsien,R.Y.(2013)Fluorescence-guided surgery with livemolecular navigation:A new cutting edge.Nat.Rev.13,653-662，该参考文献的全部内容通过引用并入本文。In one aspect, the present invention provides a method for imaging tissues or organs in a subject, comprising: administering an appropriate amount of ICG-LNP to a subject requiring imaging, and irradiating the subject with light of a suitable frequency. a subject, and acquiring images of tissues or organs of the subject by detecting light emitted by the ICG. Nguyen, Q.T., and Tsien, R.Y. (2013) Fluorescence-guided surgery with livemolecular navigation: A new cutting edge. Nat. Rev. 13, 653-662, which reference is incorporated herein by reference in its entirety.
在一些实施方式中，所述方法包括：(1)通过皮下注射、皮内注射、静脉内注射，肌肉内注射，肿瘤内注射，肿瘤周围注射的方式将包含本文提供的LNP的组合物给药于受治者，例如人类，以及(2)使用内窥镜上的近红外光/照相机或手持式探针检测来自组织内的ICG纳米颗粒的荧光，由此获取所述受治者的组织或器官的图像。In some embodiments, the method comprises: (1) administering a composition comprising LNP provided herein by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intratumoral injection, or peritumoral injection Tissue or Images of organs.
本文提供的成像方法可用于各种不同的应用，例如，(1)淋巴流和引流的绘图，(2)血管造影术，(3)检测淋巴组织，(4)前哨淋巴结绘图，(5)淋巴管结构异常检测，(6)通过改变淋巴流型或靶向癌细胞的分子检测淋巴转移癌细胞，以及(7)可视化递送包含在ICG纳米颗粒中的药物(宏观和微观/组织学)。The imaging methods provided herein can be used in a variety of different applications, for example, (1) mapping of lymphatic flow and drainage, (2) angiography, (3) detection of lymphatic tissue, (4) mapping of sentinel lymph nodes, (5) lymphatic Detection of abnormalities in tube structure, (6) detection of lymphatic metastatic cancer cells by altering lymph flow patterns or molecular targeting of cancer cells, and (7) visual delivery of drugs contained in ICG nanoparticles (macro and micro/histological).
本文提供的作为产品的ICG-LNP在仅～0.05mg/kg ICG剂量或更低的条件下有效(相对于FDA推荐的静脉内剂量：0.5-2mg/kg)(其由于强度提高了五倍并且获得了药物动力学稳定性而使剂量降低～10倍)。ICG-LNP as a product provided herein is effective at only ~0.05 mg/kg ICG dose or lower (relative to FDA recommended intravenous dose: 0.5-2 mg/kg) (which is due to the five-fold increase in strength and Pharmacokinetic stability was achieved allowing ~10-fold dose reduction).
另一方面，本发明提供一种获取受治者的组织或器官的图像的方法，所述方法包括按照单位体重计算在低于约0.5mg/kg,0.4mg/kg,0.3mg/kg,0.2mg/kg,0.1mg/kg,0.09mg/kg,0.08mg/kg,0.07mg/kg,0.06mg/kg,0.05mg/kg,0.04mg/kg,,0.03mg/kg,0.02mg/kg,或0.01mg/kg染料(例如，ICG)剂量的条件下将染料给药于所述受治者，所述染料例如，NIR染料(例如，ICG)。In another aspect, the present invention provides a method of obtaining an image of a tissue or organ of a subject, the method comprising calculating at less than about 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, 0.1mg/kg, 0.09mg/kg, 0.08mg/kg, 0.07mg/kg, 0.06mg/kg, 0.05mg/kg, 0.04mg/kg,, 0.03mg/kg, 0.02mg/kg, A dye, eg, a NIR dye (eg, ICG), is administered to the subject at a dose of 0.01 mg/kg of the dye (eg, ICG).
在一些实施方式中，所述剂量按照单位体重计算为约0.01mg/kg至约0.5mg/kg，约0.01mg/kg至约0.4mg/kg,约0.01mg/kg至约0.3mg/kg,约0.01mg/kg至约0.2mg/kg,约0.01mg/kg至约0.1mg/kg,约0.01mg/kg至约0.09mg/kg,约0.01mg/kg至约0.08mg/kg,约0.01mg/kg至约0.07mg/kg,约0.01mg/kg至约0.06mg/kg,约0.01mg/kg至约0.05mg/kg,约0.01mg/kg至约0.04mg/kg,约0.01mg/kg至约0.03mg/kg,或约0.01mg/kg至约0.02mg/kg染料(例如，ICG)剂量，包括端点值。In some embodiments, the dose is calculated from about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 0.4 mg/kg, about 0.01 mg/kg to about 0.3 mg/kg based on body weight, About 0.01 mg/kg to about 0.2 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about 0.07mg/kg, about 0.01mg/kg to about 0.06mg/kg, about 0.01mg/kg to about 0.05mg/kg, about 0.01mg/kg to about 0.04mg/kg, about 0.01mg/kg kg to about 0.03 mg/kg, or about 0.01 mg/kg to about 0.02 mg/kg dye (eg, ICG) dose, inclusive.
在一些实施方式中，剂量按照单位体重计算是约0.01mg/kg至0.5mg/kg染料(例如，ICG)剂量，包括端点值。In some embodiments, the dose is about 0.01 mg/kg to 0.5 mg/kg dye (eg, ICG) dose per body weight, inclusive.
在一些实施方式中，所述方法包括将如下量的ICG-LNP给药于受治者，所述量按照单位体重计算小于约0.5mg/kg,0.4mg/kg,0.3mg/kg,0.2mg/kg,0.1mg/kg,0.09mg/kg,0.08mg/kg,0.07mg/kg,0.06mg/kg,0.05mg/kg,0.04mg/kg,0.03mg/kg,0.02mg/kg,或0.01mg/kg ICG剂量。In some embodiments, the method comprises administering to the subject an amount of ICG-LNP that is less than about 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg per body weight /kg, 0.1mg/kg, 0.09mg/kg, 0.08mg/kg, 0.07mg/kg, 0.06mg/kg, 0.05mg/kg, 0.04mg/kg, 0.03mg/kg, 0.02mg/kg, or 0.01 mg/kg ICG dose.
在一些实施方式中，所述方法包括将如下量的ICG-LNP给药于受治者，所述量按照单位体重计算为约0.01mg/kg至约0.5mg/kg，约0.01mg/kg至约0.4mg/kg,约0.01mg/kg至约0.3mg/kg,约0.01mg/kg至约0.2mg/kg,约0.01mg/kg至约0.1mg/kg,约0.01mg/kg至约0.09mg/kg,约0.01mg/kg至约0.08mg/kg,约0.01mg/kg至约0.07mg/kg,约0.01mg/kg至约0.06mg/kg,约0.01mg/kg至约0.05mg/kg,约0.01mg/kg至约0.04mg/kg,约0.01mg/kg至约0.03mg/kg,或约0.01mg/kg至约0.02mg/kg ICG剂量，包括端点值。In some embodiments, the method includes administering ICG-LNP to the subject in an amount of about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 0.01 mg/kg based on body weight. About 0.4 mg/kg, about 0.01 mg/kg to about 0.3 mg/kg, about 0.01 mg/kg to about 0.2 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01mg/kg to about 0.08mg/kg, about 0.01mg/kg to about 0.07mg/kg, about 0.01mg/kg to about 0.06mg/kg, about 0.01mg/kg to about 0.05mg/kg kg, about 0.01 mg/kg to about 0.04 mg/kg, about 0.01 mg/kg to about 0.03 mg/kg, or about 0.01 mg/kg to about 0.02 mg/kg ICG doses, inclusive.
在一些实施方式中，所述组合物的给药途径选自全身给药和局部给药，其中，所述全身给药包括血管内注射，所述局部给药包括皮下注射、皮内注射、粘膜下注射、浆膜下注射、肿瘤内注射、肌肉内注射、口服、经鼻给药和肿瘤内注射。在本发明优选的实施方式中，在通过血管内注射的方式给药的情况下，所述组合物的量等于约0.01mg/kg体重ICG剂量至约0.5mg/kg体重ICG剂量。在局部给药本发明的组合物的情况下，所述组合物的单点注射量等于约0.1ng至约0.1mg ICG剂量。In some embodiments, the route of administration of the composition is selected from systemic administration and local administration, wherein the systemic administration includes intravascular injection, and the local administration includes subcutaneous injection, intradermal injection, mucosal injection, etc. Sub-injection, subserosal injection, intratumoral injection, intramuscular injection, oral, nasal administration and intratumoral injection. In a preferred embodiment of the present invention, in the case of administration by intravascular injection, the amount of said composition is equal to about 0.01 mg/kg body weight ICG dose to about 0.5 mg/kg body weight ICG dose. In the case of topical administration of a composition of the invention, a single point injection of the composition is equivalent to a dose of about 0.1 ng to about 0.1 mg ICG.
使用本文提供的教导，可制定不会产生很大的毒性且又完全有效治疗特定患者表现出的临床症状的有效治疗方案。这种治疗方案的制定应当涉及考虑了诸如化合物效力、相对生物利用度、患者体重、存在的副作用以及副作用的严重程度，优选的给药模式以及所选择的药剂的毒性特性之类的因素谨慎选择活性化合物。Using the teachings provided herein, effective treatment regimens can be developed that do not produce significant toxicity and yet are fully effective in treating the clinical symptoms presented by a particular patient. Development of such a treatment regimen should involve careful selection taking into account factors such as compound potency, relative bioavailability, patient body weight, presence and severity of side effects, preferred mode of administration, and the toxicity profile of the selected agent. active compound.
虽然本文已经显示并描述了本发明的优选实施方式，但是，对于本领域技术人员而言明显的是，这些实施方式仅仅是以举例说明的方式提供。在不背离本发明的条件下，本领域技术人员可做出多种改变、变化和替代。应当理解的是，对本文所述的本发明的实施方式的各种改变可在实施本发明时使用。后附的权利要求意在限定本发明的范围，其所限定的方法和结构均在这些权利要求的范围内。While preferred embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that these embodiments are provided by way of illustration only. Numerous alterations, changes, and substitutions may be made by those skilled in the art without departing from the invention. It should be understood that various modifications to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the scope of the invention be defined by the appended claims, and methods and structures defined therein are within the scope of these claims.
实施例Example
通过下文和举例说明本发明的化合物的制备的实施例进一步举例说明本发明，但不限于此。The invention is further illustrated, but not limited thereto, by the following and the examples illustrating the preparation of the compounds of the invention.
实施例1Example 1
化学试剂：吲哚菁绿(ICG；C43H47N2NaO6S2；2-[7-[3,3-二甲基-1-(4-磺丁基)苯并[e]二氢吲哚-2-亚基]庚-1,3,5-三烯-1-基]-3,3-二甲基-1-(4-磺丁基)苯并钠盐)购自Sigma-Aldrich(St.Louis，MO)。1,2-二硬脂酰基-sn-丙三基-3-磷酸胆碱(DSPC)，1,2-二硬脂酰基-sn-丙三基-3-磷酸乙醇胺-N-甲氧基-聚乙二醇-2000(DSPEmPEG2000)以及L-a-磷脂酰胆碱(Egg PC)购自Avanti Polar脂质(Alabaster，AL)。其他试剂是分析纯级别或更高级别。Chemical reagents: indocyanine green (ICG; C43 H47 N2 NaO6 S2 ; 2-[7-[3,3-dimethyl-1-(4-sulfobutyl)benzo[e]di Indoline-2-ylidene]hept-1,3,5-trien-1-yl]-3,3-dimethyl-1-(4-sulfobutyl)benzosodium salt) was purchased from Sigma - Aldrich (St. Louis, MO). 1,2-Distearoyl-sn-propanetriyl-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-propanetriyl-3-phosphoethanolamine-N-methoxy- Polyethylene glycol-2000 (DSPEmPEG2000 ) and La-phosphatidylcholine (Egg PC) were purchased from Avanti Polar lipids (Alabaster, AL). Other reagents are of analytical grade or higher.
LNP制备：对照或空LNP和ICG-LNP通过薄膜水合和声波降解法制备。简言之，将溶于CHCl3中的DSPC和溶于CHCl3:CH3OH(3:1v/v)中的DSPEmPEG2000在无菌试管中混合(9:1mol/mol)。随后在N2气和减压条件下干燥该混合物以形成薄膜，薄膜在室温条件下过夜真空干燥。用pH为7的0.9％NaCl，20mM NaHCO3缓冲液在60℃下重新水化所述薄膜持续3小时(最终脂质浓度20mM)。LNP的直径通过55℃下的15分钟的超声波浴处理降低至大约50nm至80nm。对于ICG-LNP而言，将溶于100％CH3OH的ICG加至脂质混合物中，随后干燥形成薄膜。ICG自淬灭(22)降低，并且对ICG在脂质膜中的密度进行优化(图3)。LNP和ICG-LNP的平均直径在PSS-NICOMP 380ZLS仪器(Particle Sizing Systems,Port Richey,FL)通过光子相关光谱(PCS)由粒度分析获得。ζ电位在相同的仪器上测量。ICG嵌入效率通过平衡透析由脂质结合ICG和游离ICG的分离来估算。所有实验在黑暗条件下进行，避免光线暴露。LNP preparation: Control or empty LNP and ICG-LNP were prepared by thin film hydration and sonication. Briefly, DSPC dissolved inCHCl3 and DSPEmPEG2000 dissolved inCHCl3 :CH3OH (3 :1 v/v) were mixed (9:1 mol/mol) in a sterile test tube. The mixture was then dried underN2 gas under reduced pressure to form a thin film, which was vacuum dried overnight at room temperature. The films were rehydrated with 0.9% NaCl, pH 7, 20 mM NaHCO3 buffer at 60° C. for 3 hours (final lipid concentration 20 mM). The diameter of the LNPs was reduced to approximately 50nm to 80nm by ultrasonic bath treatment at 55°C for 15 minutes. For ICG- LNP, ICG dissolved in 100% CH3OH was added to the lipid mixture followed by drying to form a thin film. ICG self-quenching (22) was reduced and the density of ICG in the lipid membrane was optimized (Figure 3). The mean diameters of LNPs and ICG-LNPs were obtained from particle size analysis by photon correlation spectroscopy (PCS) on a PSS-NICOMP 380ZLS instrument (Particle Sizing Systems, Port Richey, FL). Zeta potential was measured on the same instrument. ICG intercalation efficiency was estimated from the separation of lipid-bound and free ICG by equilibrium dialysis. All experiments were performed under dark conditions, avoiding light exposure.
吸附至LNP的ICG的90°光散射：溶于100％CH3OH的ICG与LNP以25:1至500:1的脂质-ICG摩尔比在塑料micronic管中孵育20分钟，随后用pH为7的0.9％NaCl，20mM NaHCO3的缓冲液稀释25倍以最小化ICG-脂质相互作用。随后在HitachiF-4500荧光分光光度计(Troy,MI)上测量九十度光散射。设定参数：λex/em＝660/660nm狭缝宽度ex/em＝2.5/5nm。在观察过程中在室温避光条件下保存样品。90° light scattering of ICG adsorbed to LNP: ICG dissolved in 100% CH3 OH was incubated with LNP at a lipid-ICG molar ratio of 25:1 to 500:1 in a plastic micronic tube for 20 minutes, and then washed with pH 7. Dilute 25-fold in 0.9% NaCl, 20 mM NaHCO3 buffer to minimize ICG-lipid interactions. Ninety degree light scatter was then measured on a Hitachi F-4500 spectrofluorometer (Troy, MI). Set parameters: λex/em = 660/660 nm slit widthex/em = 2.5/5 nm. During the observation process, the samples were kept at room temperature and protected from light.
荧光：使用100μL平底未处理的96孔平板(Grenier Bio-one,Monroe,NC)中的样品，在带有钨-卤素连续波灯(75W，光谱范围320nm-800nm)和激发(769±41nm)和发射(832±37nm)滤波器(Semrock,Rochester,NY)的Victor3V 1420-040多标记微孔板酶标仪(Perkin-Elmer,Waltham,MA)上进行荧光测量。Fluorescence: Use 100 μL of samples in a flat-bottomed untreated 96-well plate (Grenier Bio-one, Monroe, NC) with a tungsten-halogen continuous wave lamp (75W, spectral range 320nm-800nm) and excitation (769±41nm) Fluorescence measurements were performed on a Victor3V 1420-040 multilabel microplate reader (Perkin-Elmer, Waltham, MA) with emission (832±37 nm) filters (Semrock, Rochester, NY).
光暴露和储存稳定性：对于光暴露稳定性而言(图13)，将ICG浓度为2.0μM的游离ICG和ICG-LNP的样品暴露于顶部发光的荧光灯管持续12小时。在0小时、6小时和12小时记录荧光测量值。对于储存稳定性而言(图12)，将样品放置于黑暗中在4℃下持续多达313天。在五个不同的时间点记录游离ICG的荧光测量值，在10个不同的时间点记录ICG-LNP的荧光测量值。ICG荧光的时间依赖性衰减基于GraphPad Prism version 6.0(GraphPad Software,San Diego,CA)的指数衰减模型进行分析。数据表示为t1/2(半衰期)和k(速度常数)。Light Exposure and Storage Stability: For light exposure stability ( FIG. 13 ), samples of free ICG and ICG-LNP at an ICG concentration of 2.0 μM were exposed to top-glow fluorescent tubes for 12 hours. Fluorescence measurements were recorded at 0 hours, 6 hours and 12 hours. For storage stability (Figure 12), the samples were kept in the dark at 4°C for up to 313 days. Fluorescence measurements were recorded for free ICG at five different time points and for ICG-LNP at 10 different time points. The time-dependent decay of ICG fluorescence was analyzed based on the exponential decay model of GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). Data are expressed as t1/2 (half-life) and k (rate constant).
组织渗透深度：使用三个不同深度(0.5cm，1.0cm，1.5cm)的方块鸡胸组织体模评价ICG荧光穿过组织的检测(图2)。组织方块放置于填充有50μL的30μM游离ICG或ICG-LNP的毛细管[70μL容量，75mm长度，1.2mm内径(Fisher Scientific，Hampton，NH)]上。白光和NIR图像使用HamamatsuPhotonics K.K.(Hamamatsu,Japan)制造的定制NIR电荷耦合元件(CCD)照相机在毛细管和组织方块制备15分钟内捕获。在0-255的范围(255为最大亮度)下，所选择的区域的荧光强度平均值通过Adobe Photoshop CS4(AdobeSystems Inc.,San Jose,CA)中的分析函数获得。Tissue Penetration Depth: Detection of ICG fluorescence through tissue was evaluated using a square chicken breast tissue phantom at three different depths (0.5 cm, 1.0 cm, 1.5 cm) (Figure 2). Tissue cubes were placed on capillaries [70 μL capacity, 75 mm length, 1.2 mm inner diameter (Fisher Scientific, Hampton, NH)] filled with 50 μL of 30 μM free ICG or ICG-LNP. White light and NIR images were captured within 15 min of capillary and tissue square preparation using a custom NIR charge-coupled device (CCD) camera manufactured by Hamamatsu Photonics KK (Hamamatsu, Japan). In the range of 0-255 (255 is the maximum brightness), the average value of the fluorescence intensity of the selected area was obtained by the analysis function in Adobe Photoshop CS4 (AdobeSystems Inc., San Jose, CA).
小鼠体内NIR淋巴成像：小鼠在无菌条件下饲养，暴露于12小时光暗循环，并且在成像之前自由采食。Invivo NIR lymphatic imaging in mice: Mice were housed under sterile conditions, exposed to a 12-h light-dark cycle, and fed ad libitum before imaging.
用1.5％的异氟烷麻醉小鼠，刮除皮毛并且以仰卧位放置于IVIS Lumina IINIR CCD照相机(Perkin Elmer,Waltham,MA)下方的37℃热盘上。获取注入造影剂前的图像以确认不存在自发荧光。向两个后足的顶部皮下注射四十微升(相当于0.5nmol ICG)ICG-LNP或游离ICG。在注射之后，立即将双足放置于轻微均匀压力下。获取图像持续长达120分钟，随后在麻醉条件下通过颈脱位安乐死，小鼠安乐死之后，通过手术打开皮肤进行淋巴结成像。Mice were anesthetized with 1.5% isoflurane, shaved and placed in supine position on a 37°C hot plate beneath an IVIS Lumina IINIR CCD camera (Perkin Elmer, Waltham, MA). Acquire images prior to contrast agent injection to confirm the absence of autofluorescence. Forty microliters (equivalent to 0.5 nmol ICG) of ICG-LNP or free ICG were injected subcutaneously into the tops of both hindpaws. Immediately after the injection, place both feet under light, even pressure. Images were acquired for up to 120 minutes, followed by euthanasia by cervical dislocation under anesthesia. After euthanasia, the skin was surgically opened for lymph node imaging.
实施例2Example 2
脂质-ICG相互作用Lipid-ICG interaction
为了评价ICG和脂质之间的相互作用，将空(不含ICG)LNP与各种不同浓度的ICG一同孵育。如果溶液中的ICG分子与膜中的脂质结合，那么这会导致LNP交联和聚集，从而引起可由90度光散射变化检测的LNP表观尺寸增加以及粒度增加。在初步试验中，由鸡蛋衍生的磷脂酰胆碱(Egg PC)构成的LNP(包含混合长度的脂肪酰基链)的光散射强度随ICG浓度的增加而增加，这说明ICG诱导LNP聚集。接下来使用具有明确限定的磷脂组合物—包含两个对称C18脂肪酰基链的DSPC和DSPEmPEG2000(9:1mol/mol)—的LNP进行系统研究。使LNP中的浓度固定为10mM的脂质与不同浓度的ICG在25℃下进行相互作用持续20分钟，用缓冲液稀释25倍停止反应。对混合物进行90°光散射分析和光子相关光谱(PCS)分析以估算粒度。To evaluate the interaction between ICG and lipids, empty (without ICG) LNPs were incubated with various concentrations of ICG. If ICG molecules in solution bind to lipids in the membrane, this leads to cross-linking and aggregation of LNPs, causing an increase in the apparent size of the LNPs detectable by a change in 90 degree light scattering as well as an increase in particle size. In preliminary experiments, the light-scattering intensity of LNPs composed of egg-derived phosphatidylcholine (Egg PC) (containing fatty acyl chains of mixed lengths) increased with increasing ICG concentration, suggesting that ICG induces LNP aggregation. A systematic study was next performed using LNPs with a well-defined phospholipid composition - DSPC and DSPEmPEG2000 (9:1 mol/mol) comprising two symmetrical C18 fatty acyl chains. The lipid in LNP whose concentration was fixed at 10 mM interacted with different concentrations of ICG at 25° C. for 20 minutes, and was diluted 25-fold with buffer to stop the reaction. The mixture was subjected to 90° light scattering analysis and photon correlation spectroscopy (PCS) analysis to estimate particle size.
预见到，九十度光散射强度在颗粒处于单个，非聚集形式时较低，并且随着颗粒聚集而增强。然而，当脂质聚集体过大时，聚集体可能离开光通路或者颗粒中的电子可能不一同同相摆动并且导致颗粒内的破坏性干扰，这导致散射强度的明显下降。如图14A所示，空LNP(只有脂质)具有低散射强度，游离ICG(仅ICG)具有接近零的一致的散射强度，不论ICG的浓度是否发生改变。当将LNP和ICG(ICG-LNP)混合时，在ICG浓度较低(20-30nM)的条件下，检测到类似于空LNP对照(只有脂质)的最小光散射强度。当ICG浓度从30nM增加至70nM时，ICG-LNP的光散射强度增加大约1.5倍(图14A)。在ICG浓度为40nM时，散射强度发生较小的降低，然而，仍然显著高于ICG浓度为20-30nM的散射强度。在高浓度(100-400nM ICG)条件下，散射强度随聚集体增大而下降。图14B显示了所设想的聚集体形成的示意图，该聚集体形成导致光散射强度开始增加随后随粒度大大增加而降低。这些数据使用PCS的聚集体尺寸分析确认。如图14C所示，在20nM ICG初始浓度下，颗粒直径类似于空LNP的直径(～100nm)。表观ICG-LNP尺寸随ICG浓度从20nM增加至100nM而增加约两倍。在ICG浓度为30nM至100nM条件下表观尺寸在直径为300nm左右波动，并且随后在ICG浓度为400nM条件下增加至约400nm。在ICG浓度为30-400nM的范围内，检测到恒定直径为60-90nm的较小但独特的ICG-LNP的小聚集群(数据未显示)。It is foreseen that the ninety degree light scattering intensity is lower when the particles are in a single, non-aggregated form and increases as the particles aggregate. However, when lipid aggregates are too large, the aggregates may leave the light path or the electrons in the particle may not swing in phase and cause destructive interference within the particle, which results in a significant drop in the scattering intensity. As shown in Figure 14A, empty LNP (lipid only) had low scattering intensity, and free ICG (ICG only) had a consistent scattering intensity close to zero, regardless of changes in the concentration of ICG. When LNP and ICG (ICG-LNP) were mixed, at lower ICG concentrations (20-30 nM), minimal light scattering intensities similar to the empty LNP control (lipid only) were detected. When the concentration of ICG was increased from 30 nM to 70 nM, the light scattering intensity of ICG-LNP increased approximately 1.5-fold (Fig. 14A). At an ICG concentration of 40 nM, a smaller decrease in the scattering intensity occurred, however, still significantly higher than at an ICG concentration of 20-30 nM. At high concentrations (100-400 nM ICG), the scattering intensity decreased with aggregate size. Figure 14B shows a schematic representation of the contemplated aggregate formation that results in an initial increase in light scattering intensity followed by a decrease as the particle size increases greatly. These data were confirmed using aggregate size analysis by PCS. As shown in Figure 14C, at an initial concentration of 20 nM ICG, the particle diameter was similar to that of empty LNP (~100 nm). The apparent ICG-LNP size increased approximately two-fold with increasing ICG concentration from 20 nM to 100 nM. The apparent size fluctuates around 300 nm in diameter at ICG concentrations of 30 nM to 100 nM, and subsequently increases to about 400 nm at ICG concentrations of 400 nM. Within the range of ICG concentrations of 30-400 nM, small aggregates of smaller but distinct ICG-LNPs with a constant diameter of 60-90 nm were detected (data not shown).
综上所述，这些数据表明ICG结合至空的预先形成的LNP中存在的脂质中并且导致LNP聚集，检测为粒度明显增加，并且光散射强度不连续地增加。基于脂质浓度为10mM且ICG浓度为20-50nM，这些数据使产生最大脂质聚集和粒度明显增加的脂质-ICG摩尔比估算为200:1至500:1。Taken together, these data indicate that ICG binds to lipids present in empty preformed LNPs and leads to LNP aggregation, detected as a marked increase in particle size, and a discontinuous increase in light scattering intensity. Based on a lipid concentration of 10 mM and an ICG concentration of 20-50 nM, these data allow for estimates of lipid-ICG molar ratios of 200:1 to 500:1 that produce maximal lipid aggregation and a marked increase in particle size.
脂质-ICG相互作用对ICG荧光的影响Effect of Lipid-ICG Interaction on ICG Fluorescence
接下来，我们确定了由于ICG结合至脂质而对荧光强度产生的影响。使用脂质-ICG摩尔比125:1至25,000:1。由于ICG的自淬灭性质，用缓冲溶液稀释反应混合物20倍至线性ICG浓度范围0.01-2.0mM。如图15所示，混合物中脂质的存在使相同ICG浓度下的ICG荧光强度增加。当ICG浓度从0.01mM增加至1.0mM时，相对于对照，含有脂质的混合物的荧光强度逐渐增加。在ICG浓度为0.1mM，0.5mM和1.0mM的条件下，荧光强度分别增加了10.0倍(65,720vs.6,600)，4.3倍(261,640vs.61,360)和2.8倍(346,610vs.122,530)。在ICG浓度为2.0mM的条件下，脂质介导的ICG荧光强度的提高较小，仅仅观察到1.4倍的增加(275,820vs.199,420)。在脂质浓度固定为250μM且ICG浓度为0.5μM，1.0μM和2.0μM的条件下，这些值的等同脂质-ICG摩尔比分别为500:1，250:1和125:1(图15)。因此，表现出最大荧光强度的最优脂质-ICG摩尔比估计为125:1至500:1。该估算值与90°光散射和PCS尺寸分析收集到的数据一致。从ICG和预先形成的LNP的相互作用衍生得到的这些值用作后续ICG-LNP制备和性质研究的目标范围。Next, we determined the effect on fluorescence intensity due to ICG binding to lipids. Use a lipid-ICG molar ratio of 125:1 to 25,000:1. Due to the self-quenching nature of ICG, the reaction mixture was diluted 20-fold with buffer solution to a linear ICG concentration range of 0.01-2.0 mM. As shown in Figure 15, the presence of lipids in the mixture increased the ICG fluorescence intensity at the same ICG concentration. When the ICG concentration was increased from 0.01 mM to 1.0 mM, the fluorescence intensity of the lipid-containing mixture gradually increased relative to the control. Under the condition of ICG concentrations of 0.1mM, 0.5mM and 1.0mM, the fluorescence intensity increased by 10.0 times (65,720vs.6,600), 4.3 times (261,640vs.61,360) and 2.8 times (346,610vs.122,530), respectively. At an ICG concentration of 2.0 mM, the lipid-mediated increase in ICG fluorescence intensity was relatively small, with only a 1.4-fold increase observed (275,820 vs. 199,420). With the lipid concentration fixed at 250 μM and ICG concentrations of 0.5 μM, 1.0 μM and 2.0 μM, the equivalent lipid-ICG molar ratios for these values were 500:1, 250:1 and 125:1, respectively (Fig. 15) . Therefore, the optimal lipid-ICG molar ratio exhibiting maximum fluorescence intensity was estimated to be between 125:1 and 500:1. This estimate is consistent with data collected by 90° light scattering and PCS size analysis. These values derived from the interaction of ICG and preformed LNPs were used as target ranges for subsequent ICG-LNP preparation and property studies.
将ICG并入LNP以稳定和最大化ICG荧光Incorporation of ICG into LNPs to stabilize and maximize ICG fluorescence
不同于将溶液中的ICG加至脂质中作为缓冲溶液中的混合物，我们首先将ICG和脂质一同在有机溶剂中混合，随后除去溶剂并在缓冲液中再次水合形成插入或嵌入有ICG的LNP。ICG在吸收和发射光谱中具有显著重合(数据未显示)并且因此表现出在高浓度条件下的自淬灭可能性。因此，我们制备嵌入或并入有不同浓度(密度)的ICG的LNP，所述ICG嵌入脂质中。如果ICG密度太大，ICG分子之间非常靠近可因浓度依赖性分子相互作用而诱导自淬灭。而且，并入脂质而非暴露于水的ICG可提供比暴露于水的ICG分子更高的荧光，暴露于水会淬灭ICG荧光。评估从100:1至500:1范围内八个脂质-ICG摩尔比。图3A表示三个脂质-结合ICG样品(等于100:1，250:1和350:1mol/mol)和溶解的ICG对照的典型ICG浓度下ICG斜率的每单位荧光强度。在ICG浓度为0.01mM至2.0mM条件下，荧光强度表现出线性增加。需要注意的是，250:1线的斜率是最陡的，随后是350:1，然后是100:1和仅ICG。为了确定最优脂质-ICG摩尔比，我们评估了每个制剂的线的斜率(清楚起见，全部八条线中仅三条显示在图3A中)。Instead of adding ICG in solution to lipids as a mixture in a buffer solution, we first mixed ICG and lipids together in an organic solvent, then removed the solvent and rehydrated in buffer to form intercalated or intercalated ICG. LNP. ICG has significant overlap in absorption and emission spectra (data not shown) and thus exhibits the potential for self-quenching under high concentration conditions. Therefore, we prepared LNPs embedded or incorporated with different concentrations (density) of ICG embedded in lipids. If the ICG density is too large, the close proximity between ICG molecules can induce self-quenching due to concentration-dependent molecular interactions. Furthermore, ICG incorporated into lipids rather than exposed to water can provide higher fluorescence than ICG molecules exposed to water, which quenches ICG fluorescence. Eight lipid-ICG molar ratios ranging from 100:1 to 500:1 were evaluated. Figure 3A represents the fluorescence intensity per unit of the ICG slope at typical ICG concentrations for three lipid-bound ICG samples (equal to 100:1, 250:1 and 350:1 mol/mol) and a dissolved ICG control. Under the condition of ICG concentration of 0.01mM to 2.0mM, the fluorescence intensity showed a linear increase. Note that the slope of the 250:1 line is the steepest, followed by 350:1, then 100:1 and ICG only. To determine the optimal lipid-ICG molar ratio, we evaluated the slope of the lines for each formulation (for clarity, only three of all eight lines are shown in Figure 3A).
斜率等于每μM ICG的荧光强度。如图3B所示，由于ICG密度的降低和自淬灭，每ICG的荧光强度(斜率)随脂质-ICG摩尔比从100:1增加至250:1而增加，在脂质-ICG摩尔比为250:1的点，每ICG的荧光强度达到最大。随后，在300:1和350:1的条件下，每ICG的荧光强度降低，随后在500:1条件下显著降低。因此，我们在脂质-ICG摩尔比为250:1的条件下观察到每ICG的荧光强度峰值。The slope is equal to the fluorescence intensity per μM ICG. As shown in Figure 3B, due to the decrease in ICG density and self-quenching, the fluorescence intensity (slope) per ICG increased as the lipid-ICG molar ratio increased from 100:1 to 250:1, at the lipid-ICG molar ratio For the 250:1 point, the fluorescence intensity per ICG reaches its maximum. Subsequently, the fluorescence intensity per ICG decreased under the 300:1 and 350:1 conditions, followed by a significant decrease under the 500:1 condition. Therefore, we observed a peak fluorescence intensity per ICG at a lipid-ICG molar ratio of 250:1.
因为250:1的脂质-ICG摩尔比表现出每ICG的最大荧光强度，我们使用通过光子相关光谱(PCS)的粒度分析和通过电泳光散射(ELS)的颗粒表面电荷(ζ电位)分析表征250:1制剂。由直径为56.8±4.4nm且ζ电位为-33.1±3.1mV的颗粒的单分散群构成250:1制剂。平衡透析表明ICG合并效率为97.8±0.6％。由于ICG的并入的可重现性和ICG的几乎完全并入，选择这种制剂无需进一步纯化而用于后续的体内和体外研究。Because a lipid-ICG molar ratio of 250:1 exhibits the maximum fluorescence intensity per ICG, we characterized it using particle size analysis by photon correlation spectroscopy (PCS) and particle surface charge (zeta potential) analysis by electrophoretic light scattering (ELS). 250:1 formulation. The 250:1 formulation consisted of a monodisperse population of particles with a diameter of 56.8 ± 4.4 nm and a zeta potential of -33.1 ± 3.1 mV. Equilibrium dialysis showed an ICG incorporation efficiency of 97.8±0.6%. Due to the reproducibility of ICG incorporation and the almost complete incorporation of ICG, this preparation was chosen for subsequent in vivo and in vitro studies without further purification.
脂质并入对提高ICG暴露于光的稳定性和储存稳定性的作用Effect of Lipid Incorporation on Enhanced Light-Exposure and Storage Stability of ICG
接下来，我们评估了ICG-LNP在4℃下储存和暴露于光线下的稳定性，从而模拟临床设置的环境。如图13所示，ICG-LNP的荧光强度在暴露于光线6小时之后降低至起始值的87.6±0.5％，并且在12小时后没有发生进一步降低(t1/2＝67.5±11.8h,k＝0.011±0.002h-1)。相反，溶液中的游离ICG的荧光强度在暴露于光线6小时之后降低至其起始值的2.5±0.5％(t1/2＝0.036±0.005h,k＝19.2±2.7h-1)，这表明水溶液中的ICG光不稳定性。Next, we assessed the stability of ICG-LNP when stored at 4 °C and exposed to light, thereby mimicking the environment of a clinical setting. As shown in Figure 13, the fluorescence intensity of ICG-LNP decreased to 87.6±0.5% of the initial value after 6 hours of exposure to light, and no further decrease occurred after 12 hours (t1/2 =67.5±11.8h, k=0.011±0.002h−1 ). In contrast, the fluorescence intensity of free ICG in solution decreased to 2.5±0.5% of its initial value after 6 hours of exposure to light (t1/2 =0.036±0.005h, k=19.2±2.7h-1 ), which Indicates ICG photoinstability in aqueous solution.
为了评估长期储存稳定性，我们将ICG-LNP保存在4℃黑暗条件下并且在313天中多次测量荧光强度。如图12和表1所示，在储存ICG-LNP八个月之后，记录起始荧光强度的约78.2±2.8％(t1/2＝394天[95％CI:360,434],k＝1.76×10-3天-1[95％CI:1.60×10-3,1.93×10-3])。然而，对于缓冲液中的游离ICG而言，在储存八个月之后仅仅观察到起始ICG荧光的0.3±0.2％(t1/2＝1.19天[95％CI:1.14,1.25],k＝582×10-3天-1[95％CI:554×10-3,609×10-3])。To assess long-term storage stability, we stored ICG-LNPs in the dark at 4 °C and measured the fluorescence intensity multiple times over 313 days. As shown in Figure 12 and Table 1, after storing ICG-LNP for eight months, approximately 78.2 ± 2.8% of the initial fluorescence intensity was recorded (t1/2 = 394 days [95% CI: 360,434], k = 1.76 × 10-3 days-1 [95% CI: 1.60×10-3 , 1.93×10-3 ]). However, for free ICG in buffer, only 0.3 ± 0.2% of the initial ICG fluorescence was observed after eight months of storage (t1/2 = 1.19 days [95% CI: 1.14, 1.25], k = 582×10-3 days-1 [95% CI: 554×10-3 , 609×10-3 ]).
表1.储存稳定性动力学aTable 1. Storage stabilitykineticsa
a在4℃黑暗条件下储存。b储存八个月的样品平均值±SD(每个样品N＝2)。ct1/2(95％CI)和k(95％CI)由313天中的多个时间点(N＝10)计算。dt1/2(95％CI)和k(95％CI)由239天中的多个时间点(N＝5)计算。a Stored in the dark at 4°C.b Mean ± SD of samples stored for eight months (N=2 per sample).c t1/2 (95% CI) and k (95% CI) were calculated from multiple time points (N=10) over 313 days.d t1/2 (95% CI) and k (95% CI) were calculated from multiple time points (N=5) over 239 days.
体外增强的ICG-LNP的NIR成像NIR imaging of enhanced ICG-LNP in vitro
为了比较ICG-LNP(脂质-ICG摩尔比为250:1的制剂)和游离ICG之间的荧光信号强度，我们制备了肌肉组织厚度增加的鸡胸方块，增加的肌肉组织厚度为0.5cm、1.0cm和1.5cm(图2F)。我们用50mL 30mM的ICG(1.5nmol ICG)填充所有毛细管(长度为75mm，内径为1.2mm)(图2B)。如图2A所示，填充有ICG-LNP的毛细管的荧光强度比填充有游离ICG的毛细管的荧光强度高3.2倍(强度平均值分别为111.7vs.34.5)。当将毛细管放置于三个组织方块下方时，随着深度从左至右增加，游离ICG荧光(顶部三个组织方块)仅仅在穿过深度为0.5cm的组织方块中检测到(左上方的组织方块，强度平均值为9.0)，但不会穿过更厚的深度，ICG-LNP荧光(底部三个组织方块)在穿过0.5cm，1.0cm和1.5cm深度的组织方块中检测到(强度平均值分别为112.1，77.3，10.0)(图2C)。进一步的分析表明穿过1.5cm的肌肉组织仅可检测到毛细管中的ICG-LNP(0.5cm，1.0cm，1.5cm深度的强度平均值分别为100.9，68.0，15.2)(图2E和图2F)。To compare the fluorescence signal intensity between ICG-LNP (preparation with a lipid-ICG molar ratio of 250:1) and free ICG, we prepared chicken breast squares with increased muscle tissue thickness of 0.5 cm, 1.0 cm and 1.5 cm (Fig. 2F). We filled all capillaries (length 75 mm, inner diameter 1.2 mm) with 50 mL of 30 mM ICG (1.5 nmol ICG) (Fig. 2B). As shown in Figure 2A, the fluorescence intensity of the capillary filled with ICG-LNP was 3.2 times higher than that of the capillary filled with free ICG (intensity mean values were 111.7 vs. 34.5, respectively). When the capillary was placed below the three tissue squares, free ICG fluorescence (top three tissue squares) was detected only in tissue squares that penetrated to a depth of 0.5 cm as depth increased from left to right (top left tissue squares with an intensity mean of 9.0), but not through thicker depths, ICG-LNP fluorescence (bottom three tissue squares) was detected in tissue squares passing through 0.5cm, 1.0cm, and 1.5cm depths (intensity The mean values were 112.1, 77.3, 10.0, respectively) (Fig. 2C). Further analysis revealed that only ICG-LNP in the capillary was detectable through 1.5 cm of muscle tissue (means of intensity at 0.5 cm, 1.0 cm, and 1.5 cm depths were 100.9, 68.0, and 15.2, respectively) (Fig. 2E and Fig. 2F) .
ICG-LNP对小鼠体内NIR淋巴图像分辨率的影响Effect of ICG-LNP on the Resolution of NIR Lymphatic Image in Mice
使用稳定且最优的ICG-LNP制剂，我们在小鼠体内进行光成像实验原理的体内证明。为了比较游离ICG和ICG-LNP，我们将40μL游离形式或与LNP结合的形式的ICG(0.5nmol ICG)皮下给药于小鼠的左足或右足(图16A)。在第六分钟，只有接受ICG-LNP(而不是游离ICG)的足部表现出穿过皮肤的可检测的膝后窝结节(图16A)。仅当在皮肤下方收集到图像时，两个膝后窝淋巴结变得可检测。接受游离ICG的膝后窝淋巴结的ICG强度平均值为37.0，相对而言，用ICG-LNP处理的膝后窝淋巴结的ICG强度平均值为208.1(数据未显示)。为了进一步比较游离ICG和ICG-LNP的淋巴图像分辨率，在另一组小鼠中，我们将游离形式的ICG或LNP形式的ICG以相同的剂量给药于双足。如图16B所示，在给药后和除去皮肤之后六分钟，接受游离ICG的动物表现出ICG扩散进入血液(隐静脉)。在这种情况下，ICG在局部膝后窝结节中可清楚地检测(图16B)。相反，在用ICG-LNP处理的小鼠中，不仅膝后窝淋巴结强度高得多，而且通至腹部骨盆和生殖器/局部结节的淋巴通路也是易于看见的(图16C)。没有观察到游离ICG在该淋巴通路中的分布。Using a stable and optimal formulation of ICG-LNP, we performed an in vivo proof of principle of photoimaging experiments in mice. To compare free ICG and ICG-LNP, we administered 40 μL of ICG (0.5 nmol ICG) in free or LNP-bound form subcutaneously to the left or right paw of mice ( FIG. 16A ). At the sixth minute, only the foot receiving ICG-LNP (but not free ICG) exhibited a detectable posterior fossa nodule through the skin (Fig. 16A). Both patella lymph nodes became detectable only when images were collected just below the skin. The mean ICG intensity of the genital lymph nodes receiving free ICG was 37.0, compared with 208.1 in the genital lymph nodes treated with ICG-LNP (data not shown). To further compare the lymphatic image resolution of free ICG and ICG-LNP, in another group of mice, we administered ICG in free form or ICG in LNP form to both feet at the same dose. As shown in Figure 16B, animals receiving free ICG showed diffusion of ICG into the blood (saphenous vein) six minutes after dosing and after skin removal. In this case, ICG was clearly detectable in the focal posterior genital fossa nodules (Fig. 16B). In contrast, in mice treated with ICG-LNP, not only were the patella lymph nodes much more intense, but the lymphatic pathways to the abdominal pelvis and genital/regional nodules were also readily visible (Fig. 16C). The distribution of free ICG in this lymphatic pathway was not observed.
淋巴绘图和前哨淋巴结节检测Lymphatic mapping and sentinel lymph node detection
表2显示了在小鼠足部的皮下注射位点的下游淋巴结和血管中通过ICG-LNP和游离ICG检测到的荧光强度(膝后窝输入血管>膝后窝淋巴结>膝后窝输出血管>坐骨>髂骨肌>胃>腋窝)。在所有情况下，通过ICG-LNP检测到的强度大于通过游离ICG检测到的强度。此外，因为用ICG-LNP检测到的淋巴结的数量大于游离ICG检测到的淋巴结的数量，所以，由ICG-LNP获得更高的分辨率。这表明ICG-LNP相对于游离ICG具有提高的强度和更高的分辨率。Table 2 shows the fluorescence intensity detected by ICG-LNP and free ICG in the downstream lymph nodes and blood vessels of the subcutaneous injection site in the mouse foot (posterior genicular fossa afferent vessel>posterior genital fossa lymph node>posterior genicular fossa efferent vessel> ischia > iliac muscles > stomach > armpit). In all cases, the intensity detected by ICG-LNP was greater than that detected by free ICG. Furthermore, higher resolution was obtained from ICG-LNP because the number of lymph nodes detected with ICG-LNP was greater than that detected with free ICG. This indicates that ICG-LNP has improved intensity and higher resolution relative to free ICG.
表2.在皮下注射游离ICG和ICG-LNP之后小鼠体内所选择的淋巴结和血管中的体内荧光强度Table 2. In vivo fluorescence intensity in selected lymph nodes and blood vessels in mice after subcutaneous injection of free ICG and ICG-LNP
a在各个淋巴结或血管中检测到的最大近红外荧光(NIRF)强度(任意单位，a.u.)。平均值±SEM(N＝3-4)。NA，不可用。a The maximum near-infrared fluorescence (NIRF) intensity (arbitrary units, au) detected in each lymph node or blood vessel. Mean ± SEM (N=3-4). NA, not available.
*P值<0.05。*P value <0.05.
表3表示ICG-LNP形式中ICG剂量为皮下给药游离ICG使用的通常剂量的8％。由于本发明的脂质颗粒产生独特的ICG稳定性并使得荧光强度增强，所以脂质纳米颗粒中的ICG剂量<游离ICG所需的剂量的10％。Table 3 shows that the ICG dose in the ICG-LNP form was 8% of the usual dose used for subcutaneous administration of free ICG. Due to the unique stability of ICG produced by the lipid particles of the present invention and enhanced fluorescence intensity, the dose of ICG in the lipid nanoparticles is <10% of that required for free ICG.
表3.使用ICG-LNP和游离ICG的体内淋巴成像中典型的吲哚菁绿(ICG)皮下剂量。Table 3. Typical subcutaneous doses of indocyanine green (ICG) in in vivo lymphatic imaging using ICG-LNP and free ICG.
表4表示基于ICG是游离形式或脂质纳米颗粒形式，相等剂量的ICG表现出不同的体内药物动力学行为。ICG-LNP允许1.4倍至几乎2倍的更高的荧光强度暴露，最大强度和更快的消除速率。这些性质支持了使用ICG-LNP在体内实现荧光强度增强。Table 4 shows that equal doses of ICG exhibit different in vivo pharmacokinetic behaviors based on whether ICG is in free form or in lipid nanoparticle form. ICG-LNP allows 1.4-fold to almost 2-fold higher fluorescence intensity exposure, maximum intensity and faster elimination rate. These properties support the use of ICG-LNP to achieve fluorescence intensity enhancement in vivo.
表4.小鼠足部皮下给药之后通过皮肤检测到的第一引流淋巴结(膝后窝淋巴结)中的游离ICG和ICG-LNP的荧光强度的体内药物动力学。Table 4. In vivo pharmacokinetics of fluorescence intensity of free ICG and ICG-LNP detected through the skin in the first draining lymph node (posterior genital lymph node) after subcutaneous administration to the mouse foot.
*荧光强度＝任意单位(a.u.)* Fluorescence intensity = arbitrary unit (a.u.)
表5表示通过小鼠足部的皮下注射位点随时间推移达到的第一和第二引流淋巴节(分别为膝后窝和坐骨)中的荧光强度。两小时后，第一引流淋巴节中的信号降低了几乎一半。第二引流淋巴节中的信号(其为第一淋巴节中的信号的约70％)降低了大约25％。这说明使用ICG-LNP在体内评估起始和下游引流淋巴结的淋巴结功能的能力。Table 5 shows the fluorescence intensity in the first and second draining lymph nodes (posterior patella and ischium, respectively) reached over time through the subcutaneous injection site in the mouse foot. After two hours, the signal in the first draining lymph nodes had dropped by almost half. The signal in the second draining lymph node, which was about 70% of the signal in the first lymph node, was reduced by about 25%. This illustrates the ability to use ICG-LNP to assess lymph node function in vivo in both originating and downstream draining lymph nodes.
表5.皮下注射ICG-LNP之后来自小鼠淋巴结的体内透皮荧光强度Table 5. In vivo transdermal fluorescence intensity from mouse lymph nodes following subcutaneous injection of ICG-LNP
*荧光强度＝任意单位(a.u.)* Fluorescence intensity = arbitrary unit (a.u.)
实施例3Example 3
肿瘤前哨淋巴结检测/绘图。实施一至十次肿瘤周围深度注射并且实施一至十次肿瘤周围皮下注射。对于每次注射而言，给药0.01-10mL的ICG浓度为0.1-100μM的ICG-LNP。按摩注射位点持续大约5分钟。使用手持式、头戴式或以其他方式设置的近红外成像系统进行淋巴绘图。Tumor sentinel lymph node detection/mapping. One to ten deep peritumoral injections and one to ten peritumoral subcutaneous injections were performed. For each injection, 0.01-10 mL of ICG-LNP at an ICG concentration of 0.1-100 μM was administered. Massage the injection site for about 5 minutes. Lymphatic mapping using a handheld, head-mounted, or otherwise set-up near-infrared imaging system.
在健康个体或糖尿病、癌症或其他疾病患者体内表征淋巴功能和淋巴系统。在健康或疾病个体中，在体内的任何位置实施一至十次皮下注射。对于每次注射而言，给药0.01-10mL ICG浓度为0.1-100μM的ICG-LNP。按摩注射位点大约5分钟。使用手持式、头戴式或以其他方式设置的近红外成像系统实施淋巴绘图/成像/记录。在其他功能性特征、表型特征或描述性特征中，评价淋巴管(LV)结构，淋巴管渗透性或渗漏性，淋巴管直径，淋巴管密度，淋巴流和其他淋巴动力学参数，淋巴结(LN)形态或尺寸，淋巴结渗透性或渗漏性，淋巴结定位的变化。在使用通过淋巴检测到的荧光信号的患者体内，采用“疾病信号”：“正常信号”的比例计算表征患病和健康个体之间的淋巴差异的指数。Characterize lymphatic function and the lymphaticsystem in healthy individuals or in patients with diabetes, cancer, or other diseases . One to ten subcutaneous injections are administered anywhere in the body in healthy or diseased individuals. For each injection, 0.01-10 mL of ICG-LNP at an ICG concentration of 0.1-100 μM was administered. Massage the injection site for about 5 minutes. Perform lymphatic mapping/imaging/documentation using a handheld, head-mounted, or otherwise set-up near-infrared imaging system. Lymphatic vessel (LV) structure, lymphatic vessel permeability or leakiness, lymphatic vessel diameter, lymphatic vessel density, lymphatic flow and other lymphatic dynamic parameters, lymphatic vessel (LV) structure, lymphatic vessel (LN) Changes in morphology or size, lymph node permeability or leakiness, or lymph node location. In patients using the fluorescent signal detected through the lymph, an index characterizing the lymphatic difference between diseased and healthy individuals was calculated using the ratio of "disease signal":"normal signal".
肠道，血管和淋巴管中的淋巴管和淋巴结的内窥镜检测。使用带有皮下注射针和近红外成像能力的内窥镜，将0.01-10mL的ICG浓度为0.1-100μM的ICG-LNP注射进入肠上皮或血管内皮或淋巴管内皮。使用内窥镜近红外成像系统实施淋巴绘图。Endoscopic inspection of lymphatic vessels and lymph nodes in the intestinal tract, blood vessels and lymphatic vessels. Using an endoscope with a hypodermic needle and near-infrared imaging capability, inject 0.01-10 mL of ICG-LNP at an ICG concentration of 0.1-100 μM into the intestinal epithelium or vascular endothelium or lymphatic endothelium. Lymphatic mapping was performed using an endoscopic near-infrared imaging system.
异常肝脏功能检测。实施0.01-100mL的ICG浓度为0.1-100μM的ICG-LNP的静脉内推注或输注。使用手持式、头戴式或以其他方式设置的近红外成像系统使来自肝脏的荧光信号成像。测量来自肝脏的荧光信号的终末半衰期。与正常健康对照比较终末半衰期以评估任何肝脏机能障碍。Abnormal liver function tests. An intravenous bolus or infusion of 0.01-100 mL of ICG-LNP at an ICG concentration of 0.1-100 μM is performed. The fluorescent signal from the liver is imaged using a handheld, head-mounted, or otherwise set-up near-infrared imaging system. Measure the terminal half-life of the fluorescent signal from the liver. Terminal half-life was compared to normal healthy controls to assess any hepatic dysfunction.
转移性癌症扩散路径的检测。实施一至十次皮下、肿瘤内或肿瘤周围注射。对于每次注射而言，给药0.01-10mL的ICG浓度为0.1-100μM的ICG-LNP。如果注射位点可感知的话，按摩注射位点大约5分钟。使用手持式、头戴式内窥镜、腹腔镜或其他方式设置的近红外成像系统实施淋巴绘图。评估淋巴管和淋巴结的扩大，管堵塞或渗漏或其他与癌细胞的转移性扩散有关的特征。Detection of spread pathways in metastatic cancer. One to ten subcutaneous, intratumoral or peritumoral injections are given. For each injection, 0.01-10 mL of ICG-LNP at an ICG concentration of 0.1-100 μM was administered. If the injection site is palpable, massage the injection site for about 5 minutes. Perform lymphatic mapping using a handheld, head-mounted endoscope, laparoscope, or other near-infrared imaging system. Lymphatic vessels and lymph nodes are evaluated for enlargement, clogged or leaky tubes, or other features related to the metastatic spread of cancer cells.
血管和心血管病变检测。实施0.01-100mL ICG浓度为0.1-100μM的ICG-LNP的静脉内推注或输注。使用内窥镜，手持式，头戴式或以其他方式设置的近红外成像系统使来自血管或病理学改变的荧光信号成像以评估正常血管和心血管病变之间的差异。Vascular and cardiovascular lesion detection. An intravenous bolus or infusion of 0.01-100 mL of ICG-LNP at an ICG concentration of 0.1-100 μM is performed. Using an endoscopic, hand-held, head-mounted, or otherwise set-up near-infrared imaging system to image fluorescent signals from blood vessels or pathological changes to assess differences between normal blood vessels and cardiovascular lesions.
参考文献references
1.Nguyen QT&Tsien RY(2013)Fluorescence-guided surgery with live molecularnavigation-a new cutting edge.Nature reviews 13(9):653-662.1. Nguyen QT&Tsien RY (2013) Fluorescence-guided surgery with live molecular navigation-a new cutting edge. Nature reviews 13(9):653-662.
2.Baker KJ(1966)Binding of sulfobromophthalein(BSP)sodium and indocyanine green(ICG)by plasma alpha-1 lipoproteins.Proceedings of the Society for Experimental Biology andMedicine.Society for Experimental Biology and Medicine(New York,N.Y 122(4):957-963.2. Baker KJ (1966) Binding of sulfobromophthalein (BSP) sodium and indocyanine green (ICG) by plasma alpha-1 lipoproteins. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y 122 (4 ):957-963.
3.Mordon S,Devoisselle JM,Soulie-Begu S,&Desmettre T(1998)Indocyanine green:physicochemical factors affecting its fluorescence in vivo.Microvascular research55(2):146-152.3. Mordon S, Devoisselle JM, Soulie-Begu S, & Desmettre T (1998) Indocyanine green: physicochemical factors affecting its fluorescence in vivo. Microvascular research 55 (2): 146-152.
4.Saxena V,Sadoqi M,&Shao J(2003)Degradation kinetics of indocyanine green inaqueous solution.Journal of pharmaceutical sciences 92(10):2090-2097.4. Saxena V, Sadoqi M, & Shao J (2003) Degradation kinetics of indocyanine green inqueous solution. Journal of pharmaceutical sciences 92(10):2090-2097.
5.Zhou JF,Chin MP,&Schafer SA(1994)Aggregation and degradation of indocyaninegreen.SPIE Proc.Laser Surg.:Adv.Charact.,Ther.,Syst.IV(2128):495-505.5. Zhou JF, Chin MP, & Schafer SA (1994) Aggregation and degradation of indocyaninegreen. SPIE Proc. Laser Surg.: Adv. Charact., Ther., Syst. IV (2128): 495-505.
6.Kraft JC,Freeling JP,Wang Z,&Ho RJ(2014)Emerging research and clinicaldevelopment trends of liposome and lipid nanoparticle drug delivery systems.Journal ofpharmaceutical sciences 103(1):29-52.6. Kraft JC, Freeling JP, Wang Z, & Ho RJ (2014) Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. Journal of pharmaceutical sciences 103(1):29-52.
7.Daemen T,et al.(1997)Different intrahepatic distribution of phosphatidylglycerol andphosphatidylserine liposomes in the rat.Hepatology 26(2):416-423.7. Daemen T, et al. (1997) Different intrahepatic distribution of phosphatidylglycerol and phosphatidylserine liposomes in the rat. Hepatology 26 (2): 416-423.
8.Jeong HS,et al.(2013)The effect of mannosylation of liposome-encapsulatedindocyanine green on imaging of sentinel lymph node.Journal of liposome research.8. Jeong HS, et al. (2013) The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node. Journal of liposome research.
9.Proulx ST,et al.(2010)Quantitative imaging of lymphatic function with liposomalindocyanine green.Cancer research 70(18):7053-7062.9. Proulx ST, et al. (2010) Quantitative imaging of lymphatic function with liposomalindocyanine green. Cancer research 70(18):7053-7062.
10.Turner DC,Moshkelani D,Shemesh CS,Luc D,&Zhang H(2012)Near-infraredimage-guided delivery and controlled release using optimized thermosensitive liposomes.Pharmaceutical research 29(8):2092-2103.10. Turner DC, Moshkelani D, Shemesh CS, Luc D, & Zhang H (2012) Near-infrared image-guided delivery and controlled release using optimized thermosensitive liposomes. Pharmaceutical research 29(8):2092-2103.
11.Zhuang Y,et al.(2012)PEGylated cationic liposomes robustly augment vaccine-inducedimmune responses:Role of lymphatic trafficking and biodistribution.J Control Release159(1):135-142.11. Zhuang Y, et al. (2012) PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution. J Control Release159(1):135-142.
12.Suganami A,et al.(2012)Preparation and characterization of phospholipid-conjugatedindocyanine green as a near-infrared probe.Bioorganic&medicinal chemistry letters22(24):7481-7485.12. Suganami A, et al. (2012) Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe. Bioorganic & medicinal chemistry letters 22 (24): 7481-7485.
13.Kraft JC&Ho RJ(2014)Interactions of indocyanine green and lipid in enhancingnear-infrared fluorescence properties:the basis for near-infrared imaging in vivo.Biochemistry53(8):1275-1283.13. Kraft JC&Ho RJ(2014)Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo.Biochemistry53(8):1275-1283.
14.Hua J,et al.(2012)In vivo imaging of choroidal angiogenesis using fluorescence-labeledcationic liposomes.Molecular vision 18:1045-1054.14. Hua J, et al. (2012) In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes. Molecular vision 18:1045-1054.
15.Li X,et al.(1995)Tumor localization using fluorescence of indocyanine green(ICG)in arat model.Proc.SPIE 2389:789-797.15. Li X, et al. (1995) Tumor localization using fluorescence of indocyanine green (ICG) in arat model. Proc. SPIE 2389:789-797.
16.Philip R,Penzkofer A,Baumler W,Szeimies RM,&Abels C(1996)Absorption andfluorescence spectroscopic investigation of indocyanine green.J.Photochem.Photobiol.A:Biol.96:137-148.16. Philip R, Penzkofer A, Baumler W, Szeimies RM, & Abels C (1996) Absorption and fluorescence spectroscopic investigation of indocyanine green. J. Photochem. Photobiol. A: Biol. 96: 137-148.
17.Rajagopalan R,Uetrecht P,Bugaj JE,Achilefu SA,&Dorshow RB(2000)Stabilizationof the optical tracer agent indocyanine green using noncovalent interactions.Photochemistry andphotobiology 71(3):347-350.17. Rajagopalan R, Uetrecht P, Bugaj JE, Achilefu SA, & Dorshow RB (2000) Stabilization of the optical tracer agent indocyanine green using noncovalent interactions. Photochemistry and photobiology 71(3):347-350.
18.Engel E,et al.(2008)Light-induced decomposition of indocyanine green.Investigativeophthalmology&visual science 49(5):1777-1783.18. Engel E, et al. (2008) Light-induced decomposition of indocyanine green. Investigativeophthalmology & visual science 49 (5): 1777-1783.
19.Holzer W,et al.(1998)Photostability and thermal stability of indocyanine green.Journalof photochemistry and photobiology 47(2-3):155-164.19. Holzer W, et al. (1998) Photostability and thermal stability of indocyanine green. Journal of photochemistry and photobiology 47(2-3):155-164.
20.Sandanaraj BS,Gremlich HU,Kneuer R,Dawson J,&Wacha S(2010)Fluorescentnanoprobes as a biomarker for increased vascular permeability:implications in diagnosis andtreatment of cancer and inflammation.Bioconjugate chemistry 21(1):93-101.20. Sandanaraj BS, Gremlich HU, Kneuer R, Dawson J, & Wacha S (2010) Fluorescent nanoprobes as a biomarker for increased vascular permeability: implications in diagnosis and treatment of cancer and inflammation. Bioconjugate chemistry 21(1):93-101.
21.Proulx ST,et al.(2013)Use of a PEG-conjugated bright near-infrared dye for functionalimaging of rerouting of tumor lymphatic drainage after sentinel lymph node metastasis.Biomaterials 34(21):5128-5137.21. Proulx ST, et al. (2013) Use of a PEG-conjugated bright near-infrared dye for functional imaging of rerouting of tumor lymphatic drainage after sentinel lymph node metastasis. Biomaterials 34(21):5128-5137.
22.MacDonald RI(1990)Characteristics of self-quenching of the fluorescence oflipid-conjugated rhodamine in membranes.The Journal of biological chemistry265(23):13533-13539.22.MacDonald RI(1990)Characteristics of self-quenching of the fluorescence oflipid-conjugated rhodamine in membranes.The Journal of biological chemistry265(23):13533-13539.
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| CA (1) | CA2975853A1 (en) |
| WO (1) | WO2016123864A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106568755A (en)* | 2016-11-06 | 2017-04-19 | 浙江大学 | Near infrared laser scanning confocal microscopic imaging system |
| CN112566543A (en)* | 2018-06-14 | 2021-03-26 | 灌注科技有限公司 | System and method for automated perfusion measurement |
| CN112912087A (en)* | 2018-09-14 | 2021-06-04 | 卢卡科学株式会社 | Transplantation of mitochondria into lymphoid organs and compositions therefor |
| WO2021115070A1 (en)* | 2019-12-12 | 2021-06-17 | 深圳先进技术研究院 | Indocyanine green liposome for near-infrared region ii fluorescence detection, preparation method therefor and use thereof |
| CN115252826A (en)* | 2022-07-25 | 2022-11-01 | 四川大学华西第二医院 | Preparation method of multimodal nanoprobe for stem cell tracking |
| WO2023102877A1 (en)* | 2021-12-10 | 2023-06-15 | 深圳先进技术研究院 | Indocyanine green liposome, large-scale preparation method therefor, and application thereof |
| CN117164845A (en)* | 2023-11-02 | 2023-12-05 | 吉林大学第一医院 | Preparation method and application of polymethine nanocluster |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4349405A3 (en) | 2015-10-22 | 2024-06-19 | ModernaTX, Inc. | Respiratory virus vaccines |
| DE102017201543A1 (en)* | 2017-01-31 | 2018-08-02 | Siemens Healthcare Gmbh | Quantification of blood loss based on computed tomography with a direct-converting detector |
| US11786607B2 (en) | 2017-06-15 | 2023-10-17 | Modernatx, Inc. | RNA formulations |
| US11744801B2 (en) | 2017-08-31 | 2023-09-05 | Modernatx, Inc. | Methods of making lipid nanoparticles |
| US11911139B2 (en) | 2018-06-14 | 2024-02-27 | Perfusion Tech Aps | System and method for automatic perfusion measurement |
| EP3852728B1 (en) | 2018-09-20 | 2024-09-18 | ModernaTX, Inc. | Preparation of lipid nanoparticles and methods of administration thereof |
| CA3162183A1 (en)* | 2019-12-19 | 2021-06-24 | Morten Toft Lund | System and method for identifying blood vessels during fluorescence imaging |
| JP2024510500A (en) | 2021-03-17 | 2024-03-07 | プルーヴファーム・ライフ・ソリューションズ | Stable formulation of indocyanine green |
| BR112023018929A2 (en) | 2021-03-17 | 2023-10-10 | Provepharm Life Solutions | STABLE INDOCYANINE GREEN FORMULATIONS |
| US20240165275A1 (en) | 2021-03-17 | 2024-05-23 | Provepharm Life Solutions | Stable formulations of indocyanine green |
| CN115531558B (en)* | 2022-09-16 | 2024-07-26 | 华中科技大学 | A method for labeling and three-dimensional atlas imaging of the lymphatic vessel system in animal liver |
| CN116603078A (en)* | 2023-04-24 | 2023-08-18 | 苏州大学 | Application of indole heptamethine dyes in the preparation of oral preparations for near-infrared fluorescence imaging |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103038629A (en)* | 2010-05-31 | 2013-04-10 | 国立大学法人千叶大学 | Fluorescent probe for imaging lymph nodes |
| CN103721271A (en)* | 2013-12-17 | 2014-04-16 | 福州市传染病医院 | Multifunctional nano probe for multimodal images and photothermal therapy of liver cancer and application of multifunctional nano probe |
| CN103874482A (en)* | 2011-10-07 | 2014-06-18 | 国立大学法人鸟取大学 | Liposome composite body |
| CN103908429A (en)* | 2014-03-27 | 2014-07-09 | 深圳先进技术研究院 | Thermosensitive liposome and preparation method and application thereof |
| WO2014191467A1 (en)* | 2013-05-30 | 2014-12-04 | Bracco Imaging Spa | Fluorescent solid lipid nanoparticles composition and preparation thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0920304D0 (en)* | 2009-11-20 | 2010-01-06 | Medical Res Council | Novel liposome nanoparticles for tumour magnetic resonance imaging |
| JP2014129318A (en)* | 2012-02-23 | 2014-07-10 | Canon Inc | Indocyanine green-containing particles and photoacoustic-imaging contrast agent including the same |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103038629A (en)* | 2010-05-31 | 2013-04-10 | 国立大学法人千叶大学 | Fluorescent probe for imaging lymph nodes |
| CN103874482A (en)* | 2011-10-07 | 2014-06-18 | 国立大学法人鸟取大学 | Liposome composite body |
| WO2014191467A1 (en)* | 2013-05-30 | 2014-12-04 | Bracco Imaging Spa | Fluorescent solid lipid nanoparticles composition and preparation thereof |
| CN103721271A (en)* | 2013-12-17 | 2014-04-16 | 福州市传染病医院 | Multifunctional nano probe for multimodal images and photothermal therapy of liver cancer and application of multifunctional nano probe |
| CN103908429A (en)* | 2014-03-27 | 2014-07-09 | 深圳先进技术研究院 | Thermosensitive liposome and preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106568755A (en)* | 2016-11-06 | 2017-04-19 | 浙江大学 | Near infrared laser scanning confocal microscopic imaging system |
| CN112566543A (en)* | 2018-06-14 | 2021-03-26 | 灌注科技有限公司 | System and method for automated perfusion measurement |
| CN112912087A (en)* | 2018-09-14 | 2021-06-04 | 卢卡科学株式会社 | Transplantation of mitochondria into lymphoid organs and compositions therefor |
| US12268714B2 (en) | 2018-09-14 | 2025-04-08 | Luca Science Inc. | Transplantation of mitochondria into lymphoid organ and composition therefor |
| WO2021115070A1 (en)* | 2019-12-12 | 2021-06-17 | 深圳先进技术研究院 | Indocyanine green liposome for near-infrared region ii fluorescence detection, preparation method therefor and use thereof |
| WO2023102877A1 (en)* | 2021-12-10 | 2023-06-15 | 深圳先进技术研究院 | Indocyanine green liposome, large-scale preparation method therefor, and application thereof |
| CN115252826A (en)* | 2022-07-25 | 2022-11-01 | 四川大学华西第二医院 | Preparation method of multimodal nanoprobe for stem cell tracking |
| CN117164845A (en)* | 2023-11-02 | 2023-12-05 | 吉林大学第一医院 | Preparation method and application of polymethine nanocluster |
| CN117164845B (en)* | 2023-11-02 | 2024-02-09 | 吉林大学第一医院 | Preparation method and application of polymethine nanocluster |
| Publication number | Publication date |
|---|---|
| AU2015381503B2 (en) | 2019-08-29 |
| CA2975853A1 (en) | 2016-08-11 |
| US20160228579A1 (en) | 2016-08-11 |
| AU2015381503A1 (en) | 2017-08-24 |
| EP3253373B1 (en) | 2020-03-18 |
| CN113713124A (en) | 2021-11-30 |
| EP3253373A4 (en) | 2018-09-12 |
| JP6623479B2 (en) | 2019-12-25 |
| US10238756B2 (en) | 2019-03-26 |
| EP3253373A1 (en) | 2017-12-13 |
| JP2018504461A (en) | 2018-02-15 |
| WO2016123864A1 (en) | 2016-08-11 |
| Publication | Publication Date | Title |
|---|---|---|
| EP3253373B1 (en) | Lipid nanoparticles and uses thereof | |
| Li et al. | Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles | |
| Kraft et al. | Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo | |
| Lainé et al. | Conventional versus stealth lipid nanoparticles: formulation and in vivo fate prediction through FRET monitoring | |
| Bazylińska et al. | Nanoemulsion-templated multilayer nanocapsules for cyanine-type photosensitizer delivery to human breast carcinoma cells | |
| JP6616337B2 (en) | Peptide-containing porphyrin lipid nanovesicles | |
| WO2013125237A1 (en) | Particle containing indocyanine green, and contrast agent for photoacoustic imaging which comprises said particle | |
| Liu et al. | Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors | |
| JP2021147316A (en) | Sentinel lymph node imaging agent | |
| US9610367B2 (en) | Particle and contrast agent having the particle | |
| Plassat et al. | Sterically stabilized superparamagnetic liposomes for MR imaging and cancer therapy: pharmacokinetics and biodistribution | |
| EP2848262B1 (en) | Cell-specific targeting by nanostructured carrier systems | |
| Zhang et al. | Cisplatin and quantum dots encapsulated in liposomes as multifunctional nanocarriers for theranostic use in brain and skin | |
| US20140341813A1 (en) | Indocyanine green-containing particle and method of producing the particle | |
| Wang et al. | Evaluating the anticancer properties of liposomal copper in a nude xenograft mouse model of human prostate cancer: formulation, in vitro, in vivo, histology and tissue distribution studies | |
| MX2012009770A (en) | Liposome-encapsulated bicelles and use thereof in diluted systems. | |
| CA3117914A1 (en) | Fusogenic liposomes for selective imaging of tumor cells | |
| US9855346B2 (en) | Fluorescent dye compositions and uses thereof | |
| Charron | Fundamentals of Porphyrin-lipid J-aggregation in Nanomedicine | |
| Shi | Multifunctional thermosensitive liposomes for antitumor therapy | |
| Barker | Enzyme-Sensitive Lipid Trigger-Mediated Release of Payload from Liposomes | |
| Voigt | Liposomes for drug delivery-Surface modifications & screening for compositions at the edge of stability | |
| JP2015081230A (en) | Ph gradient method using dye complex, and dye-containing liposome | |
| Aojula | Preparation of Liposomes with Dual Fluorophores to Follow Real-Time Content Release In Vivo | |
| de Oliveira Miranda | Development of Liposomes for the Imaging of Entrapped Dyes |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20160817 |